Method for using lipoprotein associated coagulation inhibitor to treat sepsis

ABSTRACT

A method for prophylactically or therapeutically treating sepsis or septic shock is described, wherein an inhibitor to tissue factor is administered to septic patients. Additionally, a method for treating inflammation is described wherein the inhibitor is administered to pateints. This inhibitor is termed lipoprotein associated coagulation inhibitor, or commonly LACI. It is 38 kD and has 276 amino acids. LACI has now been shown to be useful for the treatment of sepsis, septic shock and inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of U.S. Ser. No. 08/224,118 filedMar. 29, 1994, which is a continuation of Ser. No. 08/020,427, filedFeb. 22, 1993 (abandoned), which is a continuation-in-part of Ser. No.07/897,135, filed Jun. 11, 1992 (abandoned). It is also acontinuation-in-part of Ser. No. 08/253,427, filed Jun. 2, 1994, whichis a continuation of Ser. No. 08/004,505 filed Jan. 13, 1993,(abandoned), which is a continuation-in-part of Ser. No. 07/891,947,filed Jun. 1, 1992 (abandoned). It is also a continuation-in-part ofSer. No. 08/270,455 filed Jul. 5, 1994, which is a continaution of Ser.No. 07/891,947, filed Jun. 1, 1992 (abandoned).

FIELD OF THE INVENTION

[0002] The present invention is a method for prophylactically andtherapeutically treating acute and chronic inflammation, sepsis andseptic shock. More specifically, it comprises administering atherapeutically effective amount of a specific protein to attenuatephysiological pathways associated with septic shock.

BACKGROUND OF THE INVENTION

[0003] Lipoprotein-associated coagulation inhibitor (LACI) is a proteininhibitor present in mammalian blood plasma. LACI is also known astissue factor (TF) inhibitor, tissue thromboplastin (Factor III)inhibitor, extrinsic pathway inhibitor (EPI) and tissue factor pathwayinhibitor (TFPI).

[0004] Blood coagulation is the conversion of fluid blood to a solid gelor clot. The main event is the conversion of soluble fibrinogen toinsoluble strands of fibrin, although fibrin itself forms only 0.15% ofthe total blood clot. This conversion is the last step in a complexenzyme cascade. The components (factors) are present as zymogens,inactive precursors of proteolytic enzymes, which are converted intoactive enzymes by proteolytic cleavage at specific sites. Activation ofa small amount of one factor catalyses the formation of larger amountsof the next, and so on, giving an amplification which results in anextremely rapid formation of fibrin.

[0005] The coagulation cascade which occurs in mammalian blood isdivided by in vitro methods into an intrinsic system (all factorspresent in the blood) and an extrinsic system which depends on theaddition of thromboplastin. The intrinsic pathway commences when thefirst zymogen, factor XII or ‘Hageman Factor’, adheres to a negativelycharged surface and in the presence of high molecular weight kininogenand prekallikrein, becomes an active enzyme, designated XIIa. Theactivating surface may be collagen which is exposed by tissue injury.Factor XIIa activates factor XI to give XIa, factor XIa activates factorIX to IXa and this, in the presence of calcium ions, a negativelycharged phospholipid surface and factor VIIIa, activates factor X. Thenegatively charged phospholipid surface is provided by platelets and invivo this serves to localize the process of coagulation to sites ofplatelet deposition. Factor Xa, in the presence of calcium ions, aplatelet-derived negatively charged phospholipid surface and a bindingprotein, factor V, activates prothrombin to give thrombin (IIa)—the mainenzyme of the cascade. Thrombin, acting on gly-arg bonds, removes smallfibrinopeptides from the N-terminal regions of the large dimericfibrinogen molecules, enabling them to polymerize to form strands offibrin. Thrombin also activates the fibrin stabilizing factor, factorXIII, to give XIIIa, a fibrinoligase, which, in the presence of calciumions strengthens the fibrin-to-fibrin links with intermolecularγ-glutamyl-ε-lysine bridges. In addition, thrombin acts directly onplatelets to cause aggregation, and release of subcellular constituentsand arachidonic acid. A further function of thrombin is to activate thecoagulation inhibitor, protein C. Factors XIIa, XIa, IXa, Xa, andthrombin are all serine proteases.

[0006] The extrinsic pathway in vivo is initiated by a substancegenerated by, or exposed by, tissue damage and termed ‘tissue factor’,interacting with Factor VII in the presence of calcium ions andphospholipid to activate factors X and IX, after which the sequenceproceeds as already described. The identity of TF is known. There isevidence that tissue factor occurs in the plasma membranes of perturbedendothelial cells of blood vessels and also in atheromatous plaques.

[0007] The two pathways described are not entirely separate because bothfactor IXa and factor XIIa in the intrinsic pathway may activate factorVII in the extrinsic pathway. There are, in addition, various feedbackloops between other factors, which enhance reaction rates. For example,thrombin (Ia) enhances the activation of both factor V and factor VIII.

[0008] Sepsis and its sequela septic shock remain among the most dreadedcomplications after surgery and in critically ill patients. The Centerfor Disease Control ranks septicemia as the 13th leading cause of deathin the United States (see MMWR, 1987, 39:31 and US Dept. of Health andHuman Services, 37:7, 1989), and the 10th leading cause of death amongelderly Americans (see MMWR, 1987, 39:777). The incidence of thesedisorders is increasing, and mortality remains high. Estimates of thetotal cost of caring for patients with septicemia range from $5 billionto $10 billion annually (see MMWR, 1987, 39:31). Death can occur in 40%to 60% of the patients. This percentage has not seen any improvementover the past 20 years. The incidence of blood borne gram-positive andgram-negative infections that can lead to septic shock occurapproximately equally.

[0009] Sepsis is a toxic condition resulting from the spread ofbacteria, or their products (collectively referred to herein asbacterial endotoxins) from a focus of infection. Septicemia is a form ofsepsis, and more particularly is a toxic condition resulting frominvasion of the blood stream by bacterial endotoxins from a focus ofinfection. Sepsis can cause shock in many ways, some related to theprimary focus of infection and some related to the systemic effects ofthe bacterial endotoxins. For example, in septacemia, bacterialendotoxins, along with other cell-derived materials, such as IL-1, IL-6and TNF, activate the coagulation system and initiate plateletaggregation. The process leads to blood clotting, a drop in bloodpressure and finally kidney, heart and lung failure.

[0010] Septic shock is characterized by inadequate tissue perfusion,leading to insufficient oxygen supply to tissues, hypotension andoliguria. Septic shock occurs because bacterial products, principallyLPS, react with cell membranes and components of the coagulation,complement, fibrinolytic, bradykinin and immune systems to activatecoagulation, injure cells and alter blood flow, especially in themicrovasculature. Microorganisms frequently activate the classiccomplement pathway, and endotoxin activates the alternate pathway.Complement activation, leukotriene generation and the direct effects ofendotoxin on neutrophils lead to accumulation of these inflammatorycells in the lungs, release of the enzymes and production of toxicoxygen radicals which damage the pulmonary endothelium and initiate theacute respiratory distress syndrome (ARDS). ARDS is a major cause ofdeath in patients with septic shock and is characterized by pulmonarycongestion, granulocyte aggregation, hemorrhage and capillary thrombi.

[0011] Activation of the coagulation cascade by bacterial endotoxinsintroduced directly into the bloodstream can result in extensive fibrindeposition on arterial surfaces with depletion of fibrinogen,prothrombin, factors V and VIII, and platelets. In addition, thefibrinolytic system is stimulated, resulting in further formation offibrin degradation products. Disseminated intravascular coagulation(DIC) is a complex coagulation disorder resulting from widespreadactivation of the clotting mechanism or coagulation cascade which, inturn, results from septicemia. Essentially, the process representsconversion of plasma to serum within the circulation system. Suchprocess represents one of the most serious acquired coagulationdisorders. Some common complications of disseminated intravascularcoagulation are severe clinical bleeding, thrombosis, tissue ischaemiaand necrosis, hemolysis and organ failure.

[0012] At the same time, as coagulation is apparently initiated byendotoxin, countervening mechanisms also appear to be activated byclotting, namely activation of the fibrinolytic system. Activated FactorXII converts plasminogen pro-activator to plasminogen activator whichsubsequently converts plasminogen to plasmin thereby mediating clotlysis. The activation of plasma fibrinolytic systems may therefore alsocontribute to bleeding tendencies.

[0013] Endotoxemia is associated with an increase in the circulatinglevels of tissue plasminogen activator inhibitor (PAI). This inhibitorrapidly inactivates tissue plasminogen activator (IPA), therebyhindering its ability to promote fibrinolysis through activation ofplasminogen to plasmin. Impairment of fibrinolysis may cause fibrindeposition in blood vessels, thus contributing to the disseminatedintravascular coagulation associated with septic shock.

[0014] Disseminated intravascular coagulation (DIC) is a coagulopathicdisorder that occurs in response to invading microorganismscharacterized by widespread deposition of fibrin in small vessels. Theinitiating cause of DIC appears to be the release of thromboplastin(tissue factor) into the circulation. During this process, there is areduction in fibrinogen and platelets, and a rise in fibrin splitproducts resulting in fibrin deposition in blood vessels. The sequenceof events that occur during DIC are described in FIG. 1. The patientseither suffer from thrombosis or hemorrhage depending on the extent ofexhaustion of the coagulation protease inhibitors during the diseaseprocess. Part of the regulation of the coagulation cascade depends onthe rate of blood flow. When flow is decreased, as it is in DIC andsepsis, the problems are magnified. DIC (clinically mild to severe form)is thought to occur with high frequency in septic shock patients andseveral other syndromes such as head trauma and burns, obstetriccomplications, transfusion reactions, and cancer. A recent abstract byXoma Corporation indicates that DIC was present on entry in 24% ofseptic patients (Martin et al., 1989, Natural History in the 1980s,Abstract No. 317, ICAAC Meeting, Dallas). Furthermore, the abstractdescribes that DIC and acute respiratory distress syndrome were thevariables most predictive of death by day 7 (risk ratios 4 and 2.3). Thecascade of events that lead to release of tissue factor into circulationand sepsis is very complex. Various cytokines are released fromactivated monocytes, endothelial cells and others; these cytokinesinclude tumor necrosis factor (TNF), interleukin 1 (IL-1) (which areknown to up-regulate tissue factor expression), interleukin 6 (IL-6),gamma interferon (IFN-γ), interleukin 8 (IL-8), and others. Thecomplement cascade is also activated as demonstrated by the rise in C3aand C5a levels in plasma of septic patients. Consequently, an agent thatwill treat coagulation without affecting the expression of tissue factoror its activity will not necessarily be effective to treat sepsis.

[0015] There are currently no satisfactory interventions for theprevention or treatment of sepsis or DIC. Heparin is the most commonlyused anticoagulant in DIC. However, it has been controversial because itcan induce bleeding and worsen the patient's condition. See, forexample, Corrigan et al., “Heparin Therapy in Septacemia withDisseminated Intravascular Coagulation. Effect on Mortality and onCorrection of Hemostatic Defects”, N. Engl. J. Med., 283:778-782 (1970);Lasch et al., Heparin Therapy of Diffuse Intravascular Coagulation(DIC)”, Thrombos. Diathes. Haemorrh., 33:105 (1974); Straub, “A CaseAgainst Heparin Therapy of Intravascular Coagulation”, Thrombos.Diathes. Haemorrh., 33:107 (1974).

[0016] Other attempts to treat sepsis using an anticoagulant have alsobeen difficult. As shown in Taylor et al., 1991, Blood, 78:364-368,warfarin and heparin are mentioned as two anticoagulants that are usedto treat DIC in sepsis, but neither are the ideal drugs. Additionally,Taylor et al. show that a new drug DEGR-Xa, a factor Xa antagonist, caninhibit DIC, however, this drug failed to block the lethal effects ofsepsis. Consequently, it is evident that an agent which may interruptthe coagulation pathway is not necessarily effective as an inhibitor ofseptic shock. Therefore, there is a need in the art for a compositionthat will inhibit the lethal effects of sepsis.

SUMMARY OF THE INVENTION

[0017] The present invention is a method for prophylactically andtherapeutically treating syndromes associated with acute or chronicinflammation where activation of Factor VII, Xa and tissue factorexpression are involved, such as sepsis and septic shock, whetheraccompanied by DIC or not. The method comprises administering aneffective amount of lipoprotein associated coagulation inhibitor (LACI).Additionally, the present invention is a method, comprisingadministering LACI, to treat a disease state in which TNF, IL-1, IL-6 orother cytokines up-regulate tissue factor. Specifically, these diseasestates include acute or chronic inflammation. Preferably, LACI isintravenously administered at a dose between 1 μg/kg and 20 mg/kg, morepreferably between 20 μg/kg and 10 mg/kg, most preferably between 1 and7 mg/kg. LACI is preferably administered with an additional agent totreat sepsis and septic shock, such as an antibiotic.

[0018] Among other things, it has been surprisingly discovered that acompound known for its anti-coagulant properties, can also attenuate theimmune response and serve as a treatment for sepsis and septic shock.This was surprising in view of the findings of Warr et al., 1990, Blood,75:1481-1489 and Taylor et al., 1991, Blood, 78:364-368.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLE

[0019]FIG. 1 shows the complex pathways involved in Sepsis and septicshock. The intrinsic and extrinsic pathways are included. Signs ofmicrovascular thrombosis include: (1) neurologic: multifocal, delerium,coma; (2) skin: focal ischemia, superficial gangrene; (3) renal:oliguria, azotemia, cortical necrosis; (4) pulmonary; acute respiratorydistress syndrome; and (5) gastrointestinal; acute ulceration. Signs ofhemorrhagic diathesis include: (1) neurologic: intracerebral bleeding;(2) skin: petechiae, ecchymoses, venepuncture oozing; (3) renal;hematuria; (4) mucous membranes: epistaxis, gingival oozing; and (5)gastrointestinal: massive bleeding.

[0020]FIG. 2 shows the inhibition of tissue factor activity by 36 dayconditioned medium (CM) and TNF induced CM.

[0021]FIG. 3 shows LACI neutralization of CM from endothelial cells.

[0022]FIG. 4 shows antibody neutralization of LACI protein.

[0023]FIGS. 5a and 5 b show pharmacokinetic profile of LACI in baboons.Open circles represent reuslts in the immunoassay and closed circlesrepresent results in the bioassay. For example, 0.5 mg/kg of LACI wasgiven as an I.V. bolus over 30 seconds to two healthy baboons. Blood wassampled from animals at +1 minute, 3, 6, 10, 20, 40, 60, 90, 120, 180,240 and 420 minutes. LACI levels in plasma were measured using bothimmunoassay and bioassay (described in text). In FIG. 5b, the linerepresents 0.7 ug/kg+10 ug/kg/min inf. 12 hr.

[0024]FIGS. 6a through 6 h show the coagulation and hematologicalresponse to LACI administration 30 minutes after the start of a two hourlethal bacterial intravenous infusion. Lines with solid circlesrepresent results obtained from treated animals and lines with “X”srepresent results obtained form control animals. A

(star) indicates a statistically significant difference (p<0.05) betweenthe control and experimental groups and an open circle represents astatistically significant (p<0.05) difference betwen times. FIG. 6ashows fibrinogen levels, FIG. 6b shows FDP levels, FIG. 6c showsplatelet levels, FIG. 6d shows WBC levels, FIG. 6e shows PT levels,Figure {circumflex over ( )}f shows APT levels, FIG. 6g shows hemtocritlevels, and FIG. 6h shows RBC levels. For example, anesthetized baboonswere challenged with a lethal dose of E. coli (˜5×10¹⁰ organisms/kg)intravenously infused over two hours. Thirty minutes after the start ofthe bacterial infusion five baboons received phosphate buffered saline(PBS; excipient control; *) and the other five received LACI in PBS (

). Blood samples were obtained from the ten baboons before the start ofthe bacterial infusion, and at 2, 4, 6, and 12 hours after the onset ofinfusion. Blood samples were assayed for fibrinogen, fibrin degradationproducts, prothrombin time, activated partial thromboplastin time, andfor hematocrit, platelet, red cell and white cell counts by standardmethods. Mean±standard error of each measurement is plotted against time(hrs.).

DETAILED DESCRIPTION OF THE INVENTION

[0025] It has now been discovered that LACI in the absence of otheranticoagulants such as heparin is effective in the prophylaxis andtreatment of sepsis. It has also been discovered that LACI alone iseffective in the prophylaxis and treatment of sepsis-associatedcoagulation disorders such as, for example, DIC. LACIinhibits/attenuates the coagulopathies and the inflammatory processassociated with acute inflammatory and septic shock.

[0026] LACI is a serum glycoprotein with a molecular weight of 38,000Kd. It is also known as tissue factor inhibitor because it is a naturalinhibitor of thromboplastin (tissue factor) induced coagulation. (U.S.Pat. Nos. 5,110,730 and 5,106,833 describe tissue factor and are herebyincorporated by reference in their entireties). LACI is a proteaseinhibitor and has 3 Kunitz domains, two of which are known to interactwith factors VII and Xa respectively, while the function of the thirddomain is unknown. Many of the structural features of LACI can bededuced because of its homology with other well-studied proteases. LACIis not an enzyme, so it probably inhibits its protease target in astoichiometric manner; namely, one of the domains of LACI inhibits oneprotease molecule. As utilized herein LACI means one or more of thethree Kunitz-type inhibitory domains of lipoprotein-associatedcoagulation inhibitor which are active in treating sepsis. The domainsmay be present on fragments of LACI or in hybrid molecules. See U.S.Pat. No. 5,106,833 regarding fragments and muteins. Preferably, Kunitzdomains 1 and/or 2 will be present. Kunitz domain 3 is not necessary foractivity.

[0027] LACI is also known as tissue factor pathway inhibitor (TFPI).This name has been accepted by the International Society on Thrombosisand Hemostasis, Jun. 30, 1991, Amsterdam. TFPI was first purified from ahuman hepatoma cell, Hep G2, as described by Broze and Miletich; Proc.Natl. Acad. Sci. USA 84:1886-1890 (1987), and subsequently from humanplasma as reported by Novotny et al., J. Biol. Chem. 264:18832-18837(1989); Chang liver and SK hepatoma cells as disclosed by Wun et al., J.Biol. Chem. 265:16096-16101 (1990). TFPI cDNA molecules have beenisolated from placental and endothelial cDNA libraries as described byWun et al., J. Biol. Chem. 263:6001-6004 (1988); Girard et al., Thromb.Res. 55, 37-50 (1989). The primary amino acid sequence of TFPI, deducedfrom the cDNA sequence, shows that TFPI contains a highly negativelycharged amino-terminus, three tandem Kunitz-type inhibitory domains, anda highly positively charged carboxyl terminus. The first Kunitz-domainof TFPI (amino acids 19 to 89 of mature TFPI and amino acids 47 to 117of pre-TFPI) is needed for the inhibition of the factor VII_(a)/tissuefactor complex and the second Kunitz-domain of TFPI (amino aicds 90 to160 of the mature protein or amino acids 118 to 188 of pre-TFPI) isresponsible for the inhibition of factor X_(a) according to Girard etal., Nature 328:518-520 (1989), while the function of the thirdKunitz-domain (amino acids 182 to 252 of mature TFPI and amino acids 210to 280 of pre-TFPI) remains unknown. See also U.S. Pat. No. 5,106,833.TFPI is believed to function in vivo to limit the initiation ofcoagulation by forming an inert, quaternary factor X_(a): TFPI: factorVII_(a): tissue factor complex. See reviews by Rapaport, Blood73:359-365 (1989), and Broze et al., Biochemistry 29:7539-7546 (1990).

[0028] Three truncated versions of LACI have been produced from E. coli.These are ala-TFPI-1-160; ala-TFPI-13-161, and ala-TFPI-22-150. Thesederivatives have production advantages and favorable solubilitycharacteristics compared to full-length ala-TFPI (ala-LACI). Thederivatives are produced at levels approximately 7-10 fold higher thanfull-length ala-LACI. Solubility of the derivatives in a physiologicalbuffer, e.g., phosphate buffered saline, is about 40 to 80-fold higherthan full-length ala-LACI. In addition, the clearance rate appearsslower for at least one of the derivatives relative to the full-lengthform. All three forms are active in factor Xa-dependent inhibition offactor VIIa/tissure factor activity. Ala-TFPI-1-160 was tested in ababoon model of sepsis and was found to promote survival. Five of eightanimals treated with the fragment survived to the 7 day endpoint, whilenone of five untreated control baboons survived.

[0029] Recombinant TFPI has been expressed as a glycosylated proteinusing mammalian cell hosts including mouse C127 cells as disclosed byDay et al., Blood 76:1538-1545 (1990), baby hamster kidney cells asreported by Pedersen et al., J. Biol. Chem. 265:16786-16793 (1990),Chinese hamster ovary cells and human SK hepatoma cells. The C127 TFPIhas been used in animal studies and shown to be effective in theinhibition of tissue factor-induced intravascular coagulation in rabbitsaccording to Day et al., supra, and in the prevention of arterialreocclusion after thrombolysis in dogs as described by Haskel et al.,Circulation 84:821-827 (1991).

[0030] Recombinant TFPI also has been expressed as a non-glycosylatedprotein using E. coli host cells yielding a highly active TFPI by invitro folding of the protein as described below in Example 1.

[0031] The cloning of the TFPI cDNA which encodes the 276 amino acidresidue protein of TFPI is further described in Wun et al., U.S. Pat.No. 4,966,852, the disclosure of which is incorporated by referenceherein.

[0032] LACI was discovered by Broze et al., 1987, PNAS (USA),84:1886-1890, and was found to inhibit Factor Xa directly, as well as toinhibit tissue factor activity by formation of an inert factorVIIa/tissue factor (IF)/Factor Xa/Ca++ inhibitor complex. It has the DNAsequence shown in U.S. Pat. No. 4,966,852 which is hereby incorporatedby reference in its entirety. A schematic diagram of the proposedmechanism for the inhibition of Factor Xa and VIIa/TF complex by LACI isshown in FIG. 1.

[0033] Coagulation occurs via two pathways: intrinsic and extrinsic. Theintrinsic and extrinsic pathways of coagulation consist of severalproteases that are activated in a series which, unless inhibited, resultin the formation of fibrin clots. LACI acts at two steps in thecoagulation cascade pathway both at the Xa and VIIa/TF level asdescribed above. The activation of tissue factor, which LACI inhibits,is a relatively early event in extrinsic pathway. (LACI has also beencalled Extrinsic Pathway Inhibitor (EPI) and tissue factor pathwayinhibitor (TFPI)). LACI inactivates Factor Xa which is a common proteasefor the extrinsic and intrinsic pathway and is downstream fromactivation of tissue factor.

[0034] The concentration of LACI in normal plasma is 100 ng/ml. A reportby Bajaj et al., 1987, J. Clin. Invest., 79:1874-1878, suggests thatLACI is synthesized in liver and endothelial cells and is consumedduring DIC in patients. Specifically, LACI values in the plasma of 15healthy volunteers ranged from 72 to 142 U/ml with a mean of 101 U/ml.Interestingly, LACI levels of 10 patients with DIC were 57±30 U/ml(p<0.001). In contrast, LACI levels of 12 patients with hepatocellulardisease were a mean of 107±33, i.e., similar to normal. Sandset et al.,1989, J. Internal Med., 225:311-316, monitored LACI plasma levels duringa 7-day observation period from patients with pneumonia (n=13), and instroke patients with infarction (n=9), and haemorrhage (n=9). Inpneumonia patients, LACI showed a weak but not significant increase inthe recovery period (p=0.068). In cerebral haemorrhage patients, LACIlevels did not consistently change, while in cerebral infarctionpatients, an increase in LACI levels was observed from day 1 to day 2(p<0.05). This latter effect was most probably due to release of tissuebound LACI by heparin and thus, was only observed in heparin-treatedpatients.

[0035] Sandset et al., 1989, Haemostasis, 19:189-195, also seriallydetermined LACI levels in 13 patients with post-operative/post-traumaticsepticemia. In the survivors (n=8), initial low LACI activity normalizedduring recovery. In the fatal cases (n=5), a progressive increase inLACI activity (maximal 30±15%) was observed until death. The increasemay be explained by a badly damaged endothelium that is releasing thetissue bound LACI into the circulation.

[0036] As utilized herein, the term “sepsis” means a toxic conditionresulting from the spread of bacterial endotoxins from a focus ofinfection.

[0037] As utilized herein, the term “sepsis-associated coagulationdisorder” means a disorder resulting from or associated with coagulationsystem activation by a bacterial endotoxin, a product of such bacterialendotoxin or both. An example of such sepsis-associated coagulationdisorder is disseminated intravascular coagulation.

[0038] The term “therapeutically-effective amount” as utilized hereinmeans an amount necessary to permit observation of activity in a patientsufficient to alleviate one or more symptoms generally associated withsepsis. Such symptoms include, but are not limited to, death, increasedheart rate, increased respiration, decreased fibrinogen levels,decreased blood pressure, decreased white cell count, and decreasedplatelet count. Preferably, a therapeutically-effective amount is anamount necessary to attenuate a decrease in fibrinogen levels in apatient being treated.

[0039] LACI Manufacture

[0040] LACI can be made and isolated by several methods. For example,cells that secrete LACI include aged endothelial cells or youngendothelial cells which have been treated with TNF for about 3 to 4days, also hepatocytes or hepatoma cells. LACI can be purified from thiscell culture by conventional methods. For example, these methods includethe chromatographic methods shown in Pedersen et al., 1990, J. ofBiological Chemistry, 265: 16786-16793, Novotny et al., 1989, J. ofBiological Chemistry, 264:18832-18837, Novotny et al., 1991, Blood,78:394-400, Wun et al., 1990, J. of Biological Chemistry, 265;16096-16101, and Broze et al., 1987, PNAS (USA), 84:1886-1890.Furthermore, LACI appears in the bloodstream and could be purified fromblood, see Pedersen et al., supra. However, that method is not suggestedor preferred because of the large quantities of blood that would berequired to obtain sufficient quantities of LACI.

[0041] LACI may be produced recombinantly as shown in U.S. Pat. No.4,966,852. For example, the cDNA for the protein can be incorporatedinto a plasmid for expression in prokaryotes or eukaryotes. U.S. Pat.No. 4,847,201,which is hereby incorporated by reference in its entirety,provides details for transforming microorganisms with specific DNAsequences and expressing them. There are many other references known tothose of ordinary skill in the art which provide details on expressionof proteins using microorganisms. Many of those are cited in U.S. Pat.No. 4,847,201, such as Maniatas, T., et al., 1982, Molecular Cloning,Cold Spring Harbor Press.

[0042] The following is an overview about transforming and expressingLACI in microorganisms. LACI DNA sequences must be isolated, andconnected to the appropriate control sequences. LACI DNA sequences areshown in U.S. Pat. No. 4,966,852 and it can be incorporated into aplasmid, such as pUNC13 or pBR3822, which are commercially availablefrom companies such as Boehringer-Mannheim. Once the LACI DNA isinserted into a vector, it can be cloned into a suitable host. The DNAcan be amplified by techniques such as those shown in U.S. Pat. No.4,683,202 to Mullis and U.S. Pat. No. 4,683,195 to Mullis et al. (LACIcDNA may be obtained by inducing cells, such as hepatoma cells (such asHepG2 and SKHep) to make LACI mRNA then identifying and isolating themRNA and reverse transcribing it to obtain cDNA for LACI.) After theexpression vector is transformed into a host such as E. coli thebacteria may be fermented and the protein expressed. Bacteria arepreferred prokaryotic microorganisms and E. coli is especiallypreferred. A preferred microorganism useful in the present invention isE. coli K-12, strain MM294 deposited with the ATCC on Feb. 14, 1984,under the provisions of the Budapest Treaty. It has accession number39607. Alternatively, LACI may be introduced into mammalian cells. Thesemammalian cells may include CHO, COS, C127, Hep G2, SK Hep, baculovirus,and infected insect cells (see also U.S. Pat. No. 4,847,201, referred toabove). See also Pedersen et al., 1990, J. of Biological Chemistry, 265:16786-16793.

[0043] Some specific details about the production of a recombinantprotein typically involves the following:

[0044] Suitable Hosts, Control Systems and Methods

[0045] First, a DNA encoding the mature protein (used here to includeall muteins); the preprotein; or a fusion of the LACI protein to anadditional sequence which does not destroy its activity or to additionalsequence cleaved under controlled conditions (such as treatment withpeptidase) to give an active protein, is obtained. If the sequence isuninterrupted by introns it is suitable for expression in any host. Ifthere are introns, expression is obtainable in mammalian or othereucaryotic systems capable of processing them. This sequence should bein excisable and recoverable form. The excised or recovered codingsequence is then placed in operable linkage with suitable controlsequences in a replicable expression vector. The vector is used totransform a suitable host and the transformed host cultured underfavorable conditions to effect the production of the recombinant LACI.

[0046] Genomic or cDNA fragments are obtained and used directly inappropriate hosts. The constructions for expression vectors operable ina variety of hosts are made using appropriate replications and controlsequences, as set forth below. Suitable restriction sites can, if notnormally available, be added to the ends of the coding sequence so as toprovide an excisable gene to insert into these vectors.

[0047] The control sequences, expression vectors, and transformationmethods are dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, or mammalian cells are presently usefulas hosts. Host systems which are capable of proper post-translationalprocessing are preferred. Accordingly, although procaryotic hosts are ingeneral the most efficient and convenient for the production ofrecombinant proteins, eucaryotic cells, and, in particular, mammaliancells are preferred for their processing capacity, for example, theability to form the proper glycosylation patterns. In addition, there ismore assurance that the native signal sequence will be recognize by themammalian host cell, thus making secretion possible, and purificationthereby easier.

[0048] Control Sequences and Corresponding Hosts

[0049] Procaryotes most frequently are represented by various strains ofE. coli. However, other microbial strains may also be used, such asbacilli, for example Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species by Bolivar, et al., 1977, Gene, 2:95. pBR322contains genes for ampicillin and tetracycline resistance, and thusprovides additional markers which can be either retained or destroyed inconstructing the desired vector. Commonly used procaryotic controlsequences are defined herein to include promoters for transcriptioninitiation, optionally with an operator, along with ribosome bindingsite sequences, which include such commonly used promoters as thebeta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., 1977, Nature, 198:1056) and the tryptophan (trp)promoter system (Goeddel, et al., 1980, Nucleic Acids Res., 8:4057) andthe λ derived P_(L) promoter and N-gene ribosome binding site(Shimatake, et al., 1981, Nature, 292:128), which has been made usefulas a portable control cassette, as set forth in U.S. Pat. No. 4,711,845,issued Dec. 8, 1987. However, any available promoter system compatiblewith procaryotes can be used.

[0050] In addition to bacteria, eucaryotic microbes, such as yeast, mayalso be used as hosts. Laboratory strains of Saccharomyces cerevisiae,Baker's yeast, are most used although a number of other strains arecommonly available. Examples of plasmid vectors suitable for yeastexpression are shown in Broach, J. R., 1983, Meth. Enz., 101:307;Stinchcomb et al., 1979, Nature, 282:39; and Tschempe et al., 1980,Gene, 10:157 and Clarke, L., et al., 1983, Meth. Enz., 101:300. Controlsequences for yeast vectors include promoters for the synthesis ofglycolytic enzymes (Hess, et al., 1968, J. Adv. Enzyme Reg., 7:149;Holland, et al., 1978, Biochemistry, 17:4900). Additional promotersknown in the art include the promoter for 3-phosphoglycerate kinase(Hitzeman, et al., 1980, J. Biol. Chem., 255:2073), and those for otherglycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and enzymesresponsible for maltose and galactose utilization (Holland, supra). Itis also believed that terminator sequences are desirable at the 3′ endof the coding sequences. Such terminators are found in the 3′untranslated region following the coding sequences in yeast-derivedgenes. Many of the vectors illustrated contain control sequences derivedfrom the enolase gene containing plasmid peno46 (Holland, M. J. et al.,1981, J. Biol. Chem., 256:1385) or the LEU2 gene obtained from YEp13(Broach, J. et al., 1978, Gene, 8:121), however, any vector containing ayeast compatible promoter, origin of replication and other controlsequences is suitable.

[0051] It is also, of course, possible to express genes encodingpolypeptides in eucaryotic host cell cultures derived from multicellularorganisms. See, for example, Tissue Culture, 1973, Cruz and Patterson,eds., Academic Press. Useful host cell lines include murine myelomasN51, VERO, HeLa cells, Chinese hamster ovary (CHO) cells, COS, C127, HepG2, SK Hep, baculovirus, and infected insect cells. Expression vectorsfor such cells ordinarily include promoters and control sequencescompatible with mammalian cells such as, for example, the commonly usedearly and later promoters from Simian Virus 40 (SV40) (Fiers, et al.,1978, Nature, 273:113), or other viral promoters such as those derivedfrom polyoma, Adenovirus 2, bovine papilloma virus, or avian sarcomaviruses, or immunoglobulin promoters and heat shock promoters. Generalaspects of mammalian cell host system transformations have beendescribed by Axel, U.S. Pat. No. 4,399,216, issued Aug. 16, 1983. It nowappears also that “enhancer” regions are important in optimizingexpression; these are, generally, sequences found upstream of thepromoter region. Origins of replication may be obtained, if needed, fromviral sources. However, integration into the chromosome is a commonmechanism for DNA replication in eucaryotes. Plant cells are also nowavailable as hosts, and control sequences compatible with plant cellssuch as the nopaline synthase promoter and polyadenylation signalsequences (Depicker, A., et al., 1982, J. Mol. Appl. Gen., 1:561) areavailable. Methods and vectors for transformation of plant cells havebeen disclosed in PCT Publication No. WO 85/04899, published Nov. 7,1985.

[0052] Host strains useful for cloning and sequencing, and forexpression of construction under control of most bacterial promotersinclude E. coli strain MM294 obtained from E. coli Genetic Stock CenterGCSC #6135. For expression under control of the P_(L)N_(RBS) promoter,E. coli strain K12 MC1000 lambda lysogen, N₇N₅₃cI857 SusP80, a straindeposited with the American Type Culture Collection (ATCC 39531), may beused. E. coli DG116, which was deposited with the ATCC (Accession No.53606) on Apr. 7, 1987, may also be used. For M13 phage recombinants, E.coli strains susceptible to phage infection, such as E. coli K12 strainDG98, can be employed. The DG98 strain has been deposited with the ATCC(ATCC 39768) on Jul. 13, 1984. Mammalian expression can be accomplishedin COS-A2 cells, COS-7, CV-1, murine myelomas N51, VERO, HeLa cells,Chinese hamster ovary (CHO) cells, COS, C127, Hep G2, SK Hep,baculovirus, and infected insect cells. Insect cell-based expression canbe in Spodoptera frugiperda.

[0053] Transformations

[0054] Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described by Cohen, S. N., 1972, PNAS(USA), 69:2110, is used for procaryotes or other cells which containsubstantial cell wall barriers. Infection with Agrobacterium tumefaciens(Shaw, C. H. et al., 1983, Gene, 23:315) is used for certain plantcells. For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, 1987, Virology,52:546 is preferred. Transformations into yeast are carried outaccording to the method of Van Solingen, P. et al., 1977, J. Bact.,130:946 and Hsiao, C. L. et al., 1979, PNAS (USA), 76:3829.

[0055] Probing mRNA by Northern Blot; Probe of cDNA or Genomic Libraries

[0056] RNA is fractionated for Northern blot by agarose slab gelelectrophoresis under fully denaturing conditions using formaldehyde,Maniatas, T., et al., 1982, Molecular Cloning, Cold Spring Harbor Press,pp. 202-203, or 10 mM methyl mercury (CH₃HgOH) (Bailey, J. M., et al.,1976, Anal. Biochem., 70:75-85; Shegal, P. B. et al., 1980, Nature,288:95-97) as the denaturant. For methyl mercury gels, 1.5% gels areprepared by melting agarose in running buffer (100 mM boric acid, 6 mMsodium borate, 10 mM sodium sulfate, 1 mM EDTA, pH 8.2), cooling to 60°C. and adding 1/100 volume of 1 M CH₃HgOH. The RNA is dissolved in0.5×running buffer and denatured by incubation in 10 mM methyl mercuryfor 10 minutes at room temperature. Glycerol (20%) and bromophenol blue(0.05%) are added for loading the samples. Samples are electrophoresedfor 500-600 volt-hr with recirculation of the buffer. Afterelectrophoresis, the gel is washed for 40 minutes in 10 mM2-mercaptoethanol to detoxify the methyl mercury, and Northern blotsprepared by transferring the RNA from the gel to a membrane filter.

[0057] cDNA or genomic libraries are screened using the colony or plaquehybridization procedure. Bacterial colonies, or the plaques for phage,are lifted onto duplicate nitrocellulose filter papers (S&S type BA-85).The plaques or colonies are lysed and DNA is fixed to the filter bysequential treatment for 5 minutes with 500 mM NaOH, 1.5 M NaCl. Thefilters are washed twice for 5 minutes each time with 5×standard salinecitrate (SSC) and are air dried and baked at 80° C. for 2 hours.

[0058] The gels for Northern blot or the duplicate filters for cDNA orgenomic screening are prehybridized at 25° to 42° C. for 6 to 8 hourswith 10 ml per filter of DNA hybridization buffer without probe (0-50%formamide, 5-6×SSC, pH 7.0, 5× Denhardt's solution(polyvinylpyrrolidone, plus Ficoll and bovine serum albumin; 1×=0.02% ofeach), 20-50 mM sodium phosphate buffer at pH 7.0, 0.2% sodium dodecylsulfate (SDS), 20 μg/ml poly U (when probing cDNA), and 50 μg/mldenatured salmon sperm DNA). The samples are then hybridized byincubation at the appropriate temperature for about 24-36 hours usingthe hybridization buffer containing kinased probe (for oligomers).Longer cDNA or genomic fragment probes were labelled by nick translationor by primer extension.

[0059] The conditions of both prehybridization and hybridization dependon the stringency desired, and vary, for example, with probe length.Typical conditions for relatively long (e.g., more than 30-50nucleotide) probes employ a temperature of 42° to 55° C. andhybridization buffer containing about 20%-50% formamide. For the lowerstringencies needed for oligomeric probes of about 15 nucleotides, lowertemperatures of about 25°-42° C., and lower formamide concentrations(0%-20%) are employed. For longer probes, the filters may be washed, forexample, four times for 30 minutes, each time at 40-55° C. with 2×SSC,0.2% SDS and 50 mM sodium phosphate buffer at pH 7, then washed twicewith 0.2×SSC and 0.2% SDS, air dried, and are autoradiographed at −70°C. for 2 to 3 days. Washing conditions are somewhat less harsh forshorter probes.

[0060] Vector Construction

[0061] Construction of suitable vectors containing the desired codingand control sequences employs standard ligation and restrictiontechniques which are well understood in the art. Isolated plasmids, DNAsequences, or synthesized oligonucleotides are cleaved, tailored, andreligated in the form desired.

[0062] Site specific DNA cleavage is performed by treating with thesuitable restriction enzyme (or enzymes) under conditions which aregenerally understood in the art, and the particulars of which arespecified by the manufacturer of these commercially availablerestriction enzymes. See, e.g., New England Biolabs, Product Catalog. Ingeneral, about 1 μg of plasmid or DNA sequence is cleaved by 1 unit ofenzyme in about 20 μl of buffer solution; in the examples herein,typically, an excess of restriction enzyme is used to insure completedigestion of the DNA substrate. Incubation times of about 1 hour to 2hours at about 37° C. are workable, although variations can betolerated. After each incubation, protein is removed by extraction withphenol/chloroform, and may be followed by ether extraction, and thenucleic acid recovered from aqueous fractions by precipitation withethanol. If desired, size separation of the cleaved fragments may beperformed by polyacrylamide gel or agarose gel electrophoresis usingstandard techniques. A general description of size separations is foundin Methods of Enzymology, 65:499-560, 1980.

[0063] Restriction cleaved fragments may be blunt ended by treating withthe large fragment of E. coli DNA polymerase I (Klenow) in the presenceof the four deoxynucleotide triphosphates (dNTPs) using incubation timesof about 15 to 25 minutes at 20° to 25° C. in 50 mM dithiothreitol (DTT)and 5-10 μM dNTPs. The Klenow fragment fills in at 5′ sticky ends butchews back protruding 3′ single strands, even though the four dNTPs arepresent. If desired, selective repair can be performed by supplying onlyone of the, or selected, dNTPs within the limitations dictated by thenature of the sticky ends. After treatment with Klenow, the mixture isextracted with phenol/chloroform and ethanol precipitated. Treatmentunder appropriate conditions with S1 nuclease results in hydrolysis ofany single-stranded portion.

[0064] Synthetic oligonucleotides may be prepared by the triester methodof Matteucci et al., 1981, J. Am. Chem. Soc., 103:3185-3191, or usingautomated synthesis methods. Kinasing of single strands prior toannealing or for labelling is achieved using an excess, e.g.,approximately 10 units of polynucleotide kinase to 1 nmole substrate inthe presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM DTT, 1-2 mM ATP.If kinasing is for labelling of probe, the ATP win contain high specificactivity 32YP.

[0065] Ligations are performed in 15-30 μl volumes under the followingstandard conditions and temperatures: 20 mM Tris-Ci pH 7.5, 10 mM MgCl₂,10 mM DTT, 33 μg/ml bovine serum albumin (BSA), 10 mM-50 mM NaCl, andeither 40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for“sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligaseat 14° C. (for “blunt end” ligation). Intermolecular “sticky end”ligations are usually performed at 33-100 μg/ml total DNA concentrations(5-100 nM total end concentration). Intermolecular blunt end ligations(usually employing a 10-30 fold molar excess of linkers) are performedat 1 μM total ends concentration.

[0066] In the vector construction employing “vector fragments”, thevector fragment is commonly treated with bacterial alkline phosphatase(BAP) in order to remove the 5′ phosphate and prevent religation of thevector. BAP digestions are conducted at pH 8 in approximately 150 mMTris, in the presence of Na²⁺ and Mg²⁺ using about 1 unit of BAP per μgof vector at 60° C. for about 1 hour. In order to recover the nucleicacid fragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors which have been double digested by additional restriction enzymedigestion of the unwanted fragments.

[0067] Modification of DNA Sequences

[0068] For portions of vectors derived from cDNA or genomic DNA whichrequire sequence modifications, site specific primer directedmutagenesis is used. This technique is now standard in the art, and isconducted using a primer synthetic oligonucleotide complementary to asingle stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Briefly, the syntheticoligonucleotide is used as a primer to direct synthesis of a strandcomplementary to the phage, and the resulting double-stranded DNA istransformed into a phage-supporting host bacterium. Cultures of thetransformed bacteria are plated in top agar, permitting plaque formationfrom single cells which harbor the phage.

[0069] Theoretically, 50% of the new plaques will contain the phagehaving, as a single strand, the mutated form: 50% will have the originalsequence. The plaques are hybridized with kinased synthetic primer at atemperature which permits hybridization of an exact match, but at whichthe mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered.

[0070] Verification of Construction

[0071] Correct ligations for plasmid construction could be confirmed byfirst transforming E. coli strain MM294, or other suitable host, withthe ligation mixture. Successful transformants are selected byampicillin, tetracycline or other antibiotic resistance or using othermarkers depending on the mode of plasmid construction, as is understoodin the art. Plasmids from the transformants are then prepared accordingto the method of Clewell, D. B. et al., 1969, PNAS (USA), 62:1159,optionally following chloramphenicol amplification (Clewell, D. B.,1972, J. Bacteriol, 110:667). The isolated DNA is analyzed byrestriction and/or sequenced by the dideoxy method of Sanger, F., etal., 1977, PNAS (USA), 74:5463 as further described by Messing et al.,1981, Nucleic Acids Res., 9:309, or by the method of Maxam et al., 1980,Methods in Enzymology, 65:499.

[0072] Purification of LACI

[0073] For purification of mammalian cell expressed LACI, the followingmethods may be used: sequential application of heparin-Sepharose, MonoQ,MonoS, and reverse phase HPLC chromatography. See Pedersen et al.,supra, Novotny et al., 1989, J. of Biological Chemistry,264:18832-18837, Novotny et al., 1991, Blood, 78:394400, Wun et al.,1990, J. of Biological Chemistry, 265:16096-16101, and Broze et al.,1987, PNAS (USA), 84:1886-1890. These references describe variousmethods for purifying mammalian produced LACI.

[0074] Additionally, LACI may be produced in bacteria, such as E. coli,and subsequently purified. Generally, the procedures shown in U.S. Pat.Nos. 4,511,502; 4,620,948; 4,929,700; 4,530,787; 4,569,790; 4,572,798;and 4,748,234 can be employed. These patents are hereby incorporated byreference in their entireties. Typically, the heterologous protein (i.e.LACI) is produced in a refractile body within the bacteria. To recoverand purify the protein, the cells are lysed and the refractile bodiesare centrifuged to separate them from the cellular debris (see U.S. Pat.No. 4,748,234 for lowering the ionic strength of the medium to simplifythe purification). Thereafter, the refractile bodies containing the LACIare denatured, at least once (typically in reducing environment), andthe protein is oxidized and refolded in an appropriate buffer solutionfor an appropriate length of time. LACI has a significant number ofcysteine residues and the procedure shown in U.S. Pat. No. 4,929,700should be relevant because CSF-1 also contains a significant number ofcysteine residues. LACI may be purified from the buffer solution byvarious chromatographic methods, such as those mentioned above for themammalian cell derived LACI. Additionally, the methods shown in U.S.Pat. No. 4,929,700 may be employed.

[0075] Administration and Formulations

[0076] LACI is administered at a concentration that is therapeuticallyeffective to treat and prevent sepsis, acute or chronic inflammation,and other diseases in which cytokines up-regulate tissue factor. Toaccomplish this goal, LACI is preferably administered intravenously.Methods to accomplish this administration are known to those of ordinaryskill in the art.

[0077] Before administration to patients, formulants may be added toLACI. A liquid formulation is preferred. In the example below, LACI wasformulated in 150 mM NaCl and 20 mM NaPO₄ at pH 7.2. However, LACI maybe formulated at different concentrations or using different formulants.For example, these formulants may include oils, polymers, vitamins,carbohydrates, amino acids, salts, buffers, albumin, surfactants, orbulking agents. Preferably carbohydrates include sugar or sugar alcoholssuch as mono, di, or polysaccharides, or water soluble glucans. Thesaccharides or glucans can include fructose, dextrose, lactose, glucose,mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin,alpha and beta cyclodextrin, soluble starch, hydroxethyl starch andcarboxymethylcelloluose, or mixtures thereof. Sucrose is most preferred.Sugar alcohol is defined as a C₄ to C₈ hydrocarbon having an —OH groupand includes galactitol, inositol, mannitol, xylitol, sorbitol,glycerol, and arabitol. Mannitol is most preferred. These sugars orsugar alcohols mentioned above may be used individually or incombination. There is no fixed limit to amount used as long as the sugaror sugar alcohol is soluble in the aqueous preparation. Preferably, thesugar or sugar alcohol concentration is between 1.0 w/v % and 7.0 w/v %,more preferable between 2.0 and 6.0 w/v %. Preferably amino acidsinclude levorotary (L) forms of carnitine, arginine, and betaine;however, other amino acids may be added. Preferred polymers includepolyvinylpyrrolidone (PVP) with an average molecular weight between2,000 and 3,000, or polyethylene glycol (PEG) with an average molecularweight between 3,000 and 5,000. It is also preferred to use a buffer inthe composition to minimize pH changes in the solution beforelyophilization or after reconstitution. Most any physiological buffermay be used, but citrate, phosphate, succinate, and glutamate buffers ormixtures thereof are preferred. Preferably, the concentration is from0.01 to 0.3 molar. Surfactants that can be added to the formulation areshown in EP Nos. 270,799 and 268,110.

[0078] Additionally, LACI can be chemically modified by covalentconjugation to a polymer to increase its circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546which are all hereby incorporated by reference in their entireties.Preferred polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH₂—CH₂)_(n)O—R where R can be hydrogen, or a protectivegroup such as an alkyl or alkanol group. Preferably, the protectivegroup has between 1 and 8 carbons, more preferably it is methyl. Thesymbol n is a positive integer, preferably between 1 and 1,000, morepreferably between 2 and 500. The PEG has a preferred average molecularweight between 1000 and 40,000, more preferably between 2000 and 20,000,most preferably between 3,000 and 12,000. Preferably, PEG has at leastone hydroxy group, more preferably it is a terminal hydroxy group. It isthis hydroxy group which is preferably activated to react with a freeamino group on the inhibitor. However, it will be understood that thetype and amount of the reactive groups may be varied to achieve acovalently conjugated PEG/IL-2 of the present invention.

[0079] Water soluble polyoxyethylated polyols are also useful in thepresent invention. They include polyoxyethylated sorbitol,polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG ispreferred. One reason is because the glycerol backbone ofpolyoxyethylated glycerol is the same backbone occurring naturally in,for example, animals and humans in mono-, di-, triglycerides. Therefore,this branching would not necessarily be seen as a foreign agent in thebody. The POG has a preferred molecular weight in the same range as PEG.The structure for POG is shown in Knauf et al., 1988, J. Bio. Chem.263:15064-15070, and a discussion of POG/IL-2 conjugates is found inU.S. Pat. No. 4,766,106, both of which are hereby incorporated byreference in their entireties.

[0080] Injectable preparations, for example, sterile injectable aqueousor oleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in water. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution, and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. A preferred injectable preparationsolution is LACI in an aqueous solution of 150 mM sodium chloride and 20mM sodium phosphate.

[0081] While LACI can be administered as the sole active anticoagulationpharmaceutical agent, it can also be used in combination with one ormore antibodies useful for treating sepsis, such as, for example,anti-endotoxin, monoclonal antibodies (endotoxin-binding Mabs) andanti-TNF products such as an anti-TNF murine Mab. LACI can also becombined with interleukin-1 receptor antagonists,bactericidal/permeability increasing (BPI) protein, immunostimulant,compounds having anti-inflammatory activity, such as PAF antagonists andcell adhesion blockers. When administered as a combination, thetherapeutic agents can be formulated as separate compositions which aregiven at the same time or different times, or the therapeutic agents canbe given as a single composition.

[0082] LACI may be given in combination with other agents which would beeffective to treat sepsis. For example, the following may beadministered in combination with LACI: antibiotics that can treat theunderlying bacterial infection; monoclonal antibodies that are directedagainst bacterial cell wall components; receptors that can complex withcytolines that are involved in the sepsis pathway; and generally anyagent or protein that can interact with cytokines or complement proteinsin the sepsis pathway to reduce their effects and to attenuate sepsis orseptic shock.

[0083] Antibiotics that are useful in the present invention includethose in the general category of: beta-lactam rings (penicillin), aminosugars in glycosidic linkage (aminoglycosides), macrocyclic lactonerings (macrolides), polycyclic derivatives of napthacenecarboxamide(tetracyclines), nitrobenzene derivatives of dichloroacetic acid,peptides (bacitracin, gramicidin, and polymyxin), large rings with aconjugated double bond system (polyenes), sulfa drugs derived fromsulfanilamide (sulfonamides), 5-nitro-2-furanyl groups (nitrofurans),quinolone carboxylic acids (nalidixic acid), and many others. Otherantibiotics and more versions of the above specific antibiotics may befound in Encyclopedia of Chemical Technology, 3rd Edition, Kirk-Othymer(ed.), Vol. 2, pages 782-1036 (1978) and Vol. 3, pages 1-78, Zinsser,MicroBiology, 17th Edition W. Joklik et al. (Eds.) pages 235-277 (1980),or Dorland's Mustrated Medical Dictionary, 27th Edition, W. B. SaundersCompany (1988).

[0084] Monoclonal antibodies that may be administered along with LACIinclude those found in PCT WO 88/03211, to Larrick et al., entitledGram-Negative Bacterial Endotoxin Blocking Monoclonal Antibodies, andU.S. Ser. No. 07/876,854, filed Apr. 30, 1992, to Larrick et al. Bothapplications disclose specific monoclonal antibodies that are useful totreat sepsis and which bind to various antigens on the E. coli bacterialcell wall. A specifically preferred monoclonal antibody is that which isproduced by hybridoma ATCC No. HB9431.

[0085] Other agents which may be combined with LACI include monoclonalantibodies directed to cytokines involved in the sepsis pathway, such asthose monoclonal antibodies directed to IL-6 or M-CSF, see U.S. Ser. No.07/451,218, filed Dec. 15, 1989 to Creasey et al. and monoclonalantibodies directed to TNF, see Cerami et al., U.S. Pat. No. 4,603,106;inhibitors of protein that cleave the mature TNF prohormone from thecell in which it was produced, see U.S. Ser. No. 07/395,253, filed Aug.16, 1989, to Kriegler et al.; antagonists of IL-1, such as shown in U.S.Ser. No. 07/517,276, filed May 1, 1990 to Haskill et al.; inhibitors ofIL-6 cytokine expression such as inhibin, as shown in U.S. Ser. No.07/494,624, filed Mar. 16, 1992, to Warren et al.; and receptor basedinhibitors of various cytokine such as IL-1. Antibodies to complement orprotein inhibitors of complement, such as CR₁, DAF, and MCP

[0086] After the liquid pharmaceutical composition is prepared, it ispreferably lyophilized to prevent degradation and to preserve sterility.Methods for lyophilizing liquid compositions are known to those ofordinary skill in the art. Just prior to use, the composition may bereconstituted with a sterile diluent (Ringer's solution, distilledwater, or sterile saline, for example) which may include additionalingredients. Upon reconstitution, the composition is preferablyadministered to subjects using those methods that are known to thoseskilled in the art.

[0087] As stated above, LACI is useful to therapeutically orprophylactically treat human patients with sepsis or septic shock, withor without DIC. Generally, people having sepsis are characterized byhigh fever (>38.5° C.) or hypothermia (<35.5° C.), low blood pressure,tachypnea (>than 20 breaths/minute), tachycardia (>than 100beats/minute), leukocytosis (>15,000 cells/mm³) and thrombocytopenia(<than 100,000 platelets/mm³) in association with bacteremia. LACIshould be administered as soon as a patient is suspected of beingseptic; presenting themselves with a greater than or equal to 20% dropin fibrinogen or appearance of fibrin split products, a rise in thepatient's temperature and the diagnosis of leukopenia, thrombocytopeniaand hypotension associated with sepsis. LACI should also be administeredwhen there is a risk of sepsis, for example, from a gunshot wound, orfrom a surgical incision. As also stated above, the preferred route isby intravenous administration. Generally, LACI is given at a dosebetween 1 μg/kg and 20 mg/kg, more preferably between 20 μg/kg and 10mg/kg, most preferably between 1 and 7 mg/kg.

[0088] Total daily dose administered to a host in single or divideddoses may be in amounts, for example, from about 2 to about 50 mg/kgbody weight daily and more usually 4 to 20 mg/kg, preferably, from about6 to about 10 mg/kg. Dosage unit compositions may contain such amountsor submultiples thereof to make up the daily dose. Lower amounts may beuseful for prophylactic or other purposes, for example, from 1 μg/kg to2 mg/kg. The amount of active ingredient that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the patient treated and the particular mode of administration.

[0089] The dosage regimen is selected in accordance with a variety offactors, including the type, age, weight, sex, diet and medicalcondition of the patient, the severity of the condition, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetic and toxicology profiles, whether a drugdelivery system is utilized and whether the compound is administered aspart of a drug combination. Thus, the dosage regimen actually employedmay vary widely and therefore may deviate from the preferred dosageregimen set forth above.

[0090] Preferably, LACI is given as a bolus dose, to increasecirculating levels by 1020 fold for 4-6 hours after the bolus dose.Continuous infusion may also be used after the bolus dose. If so, LACImay be infused at a dose between 5 and 20 μg/kg/minute, more preferablybetween 7 and 15 μg/kg/minute.

[0091] Generally, LACI may be useful for those diseases that occur dueto the up-regulation of tissue factor brought on by TNF, IL-1 or othercytokines. For example, in the examples below, LACI administration isshown to lower the IL-6 concentration. Since IL-6 is one factor that isinvolved in acute or chronic inflammation, LACI administration is usefulfor treating inflammation. Typical inflammatory conditions that can betreated by LACI include: arthritis, septic shock, reperfusion injury,inflammatory bowel disease, acute respiratory disease, trauma, and burn.

[0092] In treating chronic or acute inflammation, LACI may beadministered in the same fashion and at the same doses as in theanti-sepsis method.

[0093] The present invention will now be illustrated by reference to thefollowing examples which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

EXAMPLES Example 1

[0094] This example illustrates a method for obtaining LACI (TFPI).

[0095] Materials

[0096] Urea (sequenal grade) and Brij 35 non-ionic surfactant wereobtained from Pierce. Mixed bed resin AG501-X8 cation exchanger waspurchased from Bio Rad. Mono Q HR 5/5 and HiLoad Q Sepharose anionexchange resins, and Mono S HR 5/5 and Mono S HR 10/16 cation exchangeresins were obtained from Pharmacia. Thromboplastin reagent (SimplastinExcel) was from Organon Teknika Corp. Bovine factor X_(a) andSpectrozyme X_(a) were supplied by American Diagnostica, Inc. SDS-PAGE10-20% gradient gel was obtained from integrated Separation Systems.

[0097] Methods

[0098] Expression Vectors and Cloning Strategies

[0099] A full length human TFPI cDNA [Wun et al., J. Biol. Chem. 263,6001-6004 (1988)] was cloned into M13 mp18 phage DNA cloning vector as a1.4 Kb EcoRI fragment. Site-directed mutagenesis [Kunkel et al., Proc.Nat. Acad. Sci. USA 32, 488492 (1985)] was used to introduce an NcoIsite at the initiating ATG. The TFPI gene was then cloned as anNcoI/blunted MaeIII fragment into pMON5557 with NcoI and blunted HindIIIends resulting in the new vector pMON9308. MaeIII site is 15 bpdownstream from the stop codon in the TFPI cDNA. The expression vectorcontained the recA promoter, a translational enhancer element andribosome binding site derived from the gene 10 leader of bacteriophageT7 as described by Olins and Rangwala, J. Biol. Chem. 264, 16973-16976(1989), and the T7 transcription terminator. This plasmid also containsan irrelevant sequence, i.e. the bST gene (bovine somatotropin).

[0100] The NcoI/NsiI fragment of pMON9308 was then replaced by asynthetic DNA fragment designed to (1) introduce an alanine encodingcodon at the second position, (2) increase the A-T richness of the 5′portion of the gene, and (3) improve E. coli codon usage. Fouroligonucleotides, two for each strand, were used. All base substitutions(indicated in upper case), are silent changes. ECTFPI 2 and 3 were 5′phosphorylated [Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982)]. ECTFPI1 and 2 and ECTFPI 3 and 4 were annealed in the kinase buffer byincubating for 5 minutes at 70° C. and slow-cooling to room temperature.These fragments were cloned into pMON9308 which had been digested withNcoI/NsiI. PCR amplification was used to introduce a HindIII site aswell as a TAA termination codon at the 3′ end of the TFPI gene. The PCRprimers TPFIterm and TPFIterm 2 are shown below. The TFPI gene was thenmoved as a NCOI/HindIII fragment into pMON5766. The resultant plasmidwas pMON6870. N c o           ECTFPI 1 lcatggctgattctgaAgaagatgaagaacaTacTa     cgactaagactTcttctacttcttgtAtgAtaatagtgA             ECTFPI 2                         N                          s             ECTFPI3     i                          lttatcacTgatacTgaACtgccaccGctgaaactGatgca     ctatgActTGacggtggCgactttgaCt             ECTFPI 4                     HindIII TFPIterm:   ataaca[aagctt]acatatttt                     NcoI TFPIterm2:   atatat[ccatgg]ctgattct

[0101] pMON6870 was digested with Bg1II/HindIII. This fragment,containing the expression cassette, was cloned into pMON6710 [Obukowiczet al., Biochemistry 29, 9737-9745 (1990)] which had been digested withBg1II/HindIII. The resultant plasmid, pMON6875, includes the tacpromoter, G10 leader from bacteriophage T7, met-ala TFPI, and the p22transcriptional terminator. The plasmids were transformed into MON105(rpoD +rpoH358) containing F′ from JM101 for the expression of TFPIprotein.

[0102] Fermentation

[0103] Ten liter fermentations were run in M9 minimal salts mediasupplemented with 20 g/l casamino acids in Biostad E fermentors (B.Braun). Fermentations were run at a temperature of 37° C., 1000 rpmagitation, an air flow rate of 15 l/min and 10 psi backpressure. pH wascontrolled at 7.0 with ammonium hydroxide. Residual glucoseconcentration in the fermentation broth was automatically controlled at1.0+/−0.1 g/l. At an optical density of 46.0 at 550 nm, the temperaturewas shifted from 37° C. to 30° C. and isopropyl β-Dthiogalactopyranoside (IPTG) was added to the fermentor to a finalconcentration of 1.0 mM. The culture was harvested four hourspost-induction by concentration in an Amicon DC10L concentrator followedby centrifugation in a Beckman J2-21 centrifuge. The 10-literfermentation yield 335-456 g (average of 376+/−46 g, n=6) wet weight ofcell paste. The cell paste was frozen at −80° C. for further processinghereinbelow.

[0104] Isolation of Inclusion Bodies

[0105] Frozen E. coli cell paste was resuspended in cold Milli-Q waterat a concentration of 75 g/l. The cells were thoroughly dispersed with ahomogenizer (Ultra-Turrax model SD45) for 30 minutes on ice. The cellswere mechanically lysed by three passes through the Manton-Gaulinhomogenizer (model 15M-8TA) at 12,000 psi. Inclusion bodies werecentrifuged in the Sorvall RC-2B centrifuge in the GSA rotor at 10,000rpm (16,270×g) for 20 minutes. The supernatant was discarded. Theinclusion body pellets were collected, resuspended in 1 liter of coldMilli-Q water and dispersed with the Ultra-Turrax homogenizer for 30minutes on ice. The inclusion bodies were cycled through theManton-Gaulin homogenizer two more times on ice. Inclusion bodies werepelleted in the Sorvall RC-2B centrifuge as before. Approximately 60 mgof inclusion bodies were collected for every gram of E. coli cellslysed. The inclusion bodies were stored at −80° C.

[0106] Buffer preparation

[0107] All the buffers used for sulfonation and refolding of E. coliTFPI contained high concentrations of urea. Urea solutions were treatedwith Bio-Rad mixed bed resin AG501-X8 at room temperature for at least20 minutes and filtered through 0.2 μm filter before mixing withbuffers. All the solutions used for chromatography were 0.2 μm filteredand sonicated under house vacuum for about 10 minutes.

[0108] Sulfonation of Inclusion Bodies

[0109] One gm of inclusion bodies (wet weight) was dispersed in 40 ml ofa solution containing 50 mM Tris/HCl, pH 8, and 7.5 M urea byhomogenization and vortexing. After the inclusion bodies were largelydissolved, 800 mg of sodium sulfite was added and the mixture was shakenat room temperature for 30 minutes. Then, 400 mg of sodium dithionite or120 mg of sodium tetrathionate was added and the mixture was shaken at4° C. overnight. The solution dialyzed against 800 ml of a solutioncontaining 20 mM Tris/HCl, pH 8, and 6 M urea for more than 5 hours at4° C. using a Spectrapor #2 membrane. The dialyzed solution wascentrifuged at 48,400×g for 1 hour, filtered through a 0.2 μm filter,divided into aliquots, and stored at −80° C.

[0110] Anion-Exchange Chromatography of Sulfonated TFPI

[0111] On a small scale, the sulfonated and dialyzed inclusion bodieswere fractionated on a Mono Q HR5/5 anion exchange column. The columnwas pre-equilibrated in Q-buffer (20 mM Tris/HCl, pH 8, 6 M urea, 0.01%Brij 35 non-ionic surfactant) containing 0.15 M NaCl. Two ml ofsulfonated inclusion bodies were loaded onto the column. The column waswashed with 15 ml of the equilibration buffer and eluted with a 30-mlgradient (0.15-0.4 M NaCl) in Q-buffer. Fractions of 1 ml werecollected. On a larger scale, 40 ml of sulfonated sample (equivalent to0.56 g of wet weight inclusion body) was loaded onto a HiLoad QSepharose 16/10 anion exchange column pre-equilibrated in Q-buffercontaining 0.15 M NaCl. The column was washed with 240 ml ofequilibration buffer and then eluted with a 396-ml gradient (0.15-0.4 MNaCl) in Q-buffer. Nine ml fractions were collected. Bothchromatographies were carried out on a Pharmacia FPLC system at roomtemperature.

[0112] Refold of Sulfonated TFPI

[0113] The sulfonated, full-length TFPI pool from anion-exchangechromatography was diluted to an absorbance of 0.07 O.D. units at 280 nmwith Q-buffer containing 0.3 M NaCl. Solid L-cysteine was added to afinal concentration of 2 mM. The solution was incubated at roomtemperature for 24 hours, diluted 1:1 with water, 1 mM L-cysteine wasadded, incubated at room temperature for another 24 hours and thenincubated at 4° C. for up to 4 to 8 days. pH was maintained at 8.5 byaddition of 50 mM Tris. Mono S Chromatography of refold mixture Inanalytical runs, 2 ml refold mixture was loaded onto a Mono S HR 5/5cation exchange column pre-equilibrated in S-buffer (20 mM sodiumphosphate, pH 6.4, 6 M urea). The column was washed with 10 ml of theequilibration buffer and eluted with a 70-ml gradient consisting of0-0.7 M NaCl in S-buffer. One-ml fractions were collected. Inpreparative runs, the refold mixture was acidified to pH 4.5,concentrated 75-fold, and loaded onto a Mono S HR10/16 anion exchangecolumn pre equilibrated in S-buffer containing 0.3 M NaCl. The columnwas washed with 15-column volumes of the equilibration buffer and elutedwith a 0.3-0.5 M NaCl gradient in S-buffer.

[0114] Tissue Factor-Induced Coagulation Time Assay

[0115] Conventional coagulation time assay was performed using aFibrometer (Becton Dickinson) clot timer. Ninety μl of human pooledplasma was mixed with 10 μl of TFPI sample or control buffer in the wellat 37° C. for 1 min and 0.2 ml of tissue factor (Simplastin Excel,diluted 1:60 into a solution containing 75 mM NaCl, 12.5 mM CaCl₂, and0.5 mg/ml bovine serum albumin) was added to initiate the clottingreaction.

[0116] Amidolytic Assay of factor X_(a) Inhibitory Activity

[0117] Inhibitory activity against bovine factor X_(a) of TFPI sampleswere assayed by conventional amidolysis of Spectrozme X_(a) as describedpreviously by Wun et al., J. Biol. Chem. 265, 16096-16101 (1990) exceptthat the assay buffer consisted of 0.1 M Tris/HCl, pH 8.4, and 0.1%Triton X-100 non-ionic surfactant.

[0118] Protein Determination

[0119] The concentration of protein was determined by absorbance at 280nm and by quantitative amino acid analysis after HCl/vapor phasehydrolysis at 110° C. for 24 hours.

[0120] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE)

[0121] Daiichi precasted 10-20% gradient gels were used for SDS-PAGE.Samples are either unreduced and not boiled or reduced in 3.3%2-mercaptoethanol and boiled for 3 minutes before electrophoresis. Thegels were stained by Coomassie blue.

[0122] Expression of TFPI in E. coli

[0123] Three vectors were constructed and used for expression of TFPI inE. coli. The first construct, pMON9308, which contained the originalhuman TFPI cDNA sequence (except the initiating ATG) and the rec Apromoter, achieved a very low level of expression (<0.5% of total cellprotein). The second construct, pMON6870, which was similar to the firstbut was altered by introducing an alanine at the second position, byincreasing the A-T richness of the 5′-end and by improving E. coli codonusage, did not significantly raise the expression level. The thirdconstruct pMON6875, which was similar to the second but used a tacpromoter, achieved an expression level of approximately 5-10% of totalcell protein and was used for further tests herein. The majority of TFPI(>90%) appeared to be sequestered in inclusion bodies.

[0124] Sulfonation of Inclusion Body and Purification of Full-LengthSulfonated TFPI

[0125] In initial tests, it was found that the E. coli lysate or theisolated inclusion bodies contained very little TFPI activity asmeasured by anti-factor X_(a) and by tissue factor-induced coagulationtime assays. Refolding of TFPI by reduction/re-oxidation and bysulfonation/disulfide interchange of the crude, solubilized inclusionbodies resulted in very low recovery of activity. Therefore, attemptswere made to purify TFPI prior to refolding step, by sulfonationfollowed by anion exchange chromatography, taking advantage of the 18added negatively charged groups on the sulfonated TFPI. The sulfonatedinclusion bodies were first fractionated on an analytical Mono Q HR5/5anion exchange column. The flow-through and early gradient fractionscontained much of the contaminants E. coli protein and truncated TFPIprotein (the latter are lower in molecular weight and areimmuno-reactive against anti-TFPI-Ig). The full-length TFPI-S-sulfonateeluted at about 0.28 M NaCl. The fractionation of sulfonated inclusionbodies was scaled up 20 times using a Hiload Q Sepharose 16/10 anionexchanger. The chromatogram appeared somewhat different from that fromMono Q but the fractionation of the full-length TFPI-S-sulfonateappeared comparable as judged from SDS-PAGE.

[0126] Refold of TFPI-S-Sulfonate

[0127] Sulfonated TFPI underwent spontaneous refolding and oxidationupon mixing with a suitable concentration of L-cysteine. The efficiencyof refold as reflected in the increase of TFPI activity varies widelydepending on the refold conditions. Numerous refold conditions werecompared and optimized in terms of temperature, pH, urea, L-cysteine andprotein concentration. A 2-stage refold process appeared to be the best.In the first stage, the full-length TFPI-S-sulfonate pool was adjustedto an absorbance at 280 nm of 0.07 O.D. units, 2 mM of fresh L-cysteinewas added, and the mixture was incubated at room temperature for 24hours. During this period, the TFPI activity increased from 0 to about12% of full-length SK hepatoma TFPI which served as a standard forcomparison. In the second stage, the solution was diluted 1:1 withwater, and fresh L-cysteine was added to a final concentration of 1 mM.The mixture was incubated at room temperature for 24 hours, during whichtime the specific activity increased about 2 fold to about 30% that ofSK Hepatoma TFPI. The solution was then left at 4° C. for several daysduring which time the TFPI activity increased.

[0128] Fractionation of Refold Mixture by Mono S Chromatography

[0129] The specific activity of the refold mixture was lower than thepurified mammalian SK TFPI which suggests that the former may containboth correctly folded and misfolded molecules or only partially activemisfolded molecules. The refold mixture was fractionated on ananalytical Mono S cation exchange column. When the UV-absorbingfractions were analyzed for TFPI activity, the highest specific activitywas associated with a sharp peak (fraction 52) eluted at 0.52 M NaCl.All the other fractions had a specific activity less than 30% that offraction 52. SDS-PAGE analysis showed that fraction 52 contained a sharpband and all other fractions, together with pre-column refold mixture,consisted of diffuse, multiple bands under nonreducing condition. Thediffuse bands are apparently mainly full-length TFPI in various foldedforms since they become sharp-banded upon reduction (see the last twolanes on the right). By making the gradient more shallow, the resolutionof the peaks become better and all the protein peaks appeared to eluteat lower NaCl concentrations Further, it was possible to wash out themajority of the low-activity peaks with 10 column volumes of 0.3 M NaClbefore eluting the active peak with a shadow gradient.

[0130] Based on the above results, the chromatography was scaled upusing a Mono S HR10/16 cation exchange column. The column was washedwith 15 column volumes of 0.3 M NaCl which essentially washed out alllow activity peaks. Afterwards, a shadow gradient eluted a peak ofprotein that contained the active TFPI. SDS-PAGE analysis shows that thepeak gave a sharp band under either reducing or non-reducing conditions.The reduced and boiled protein migrated somewhat slower in SDS-PAGE.

[0131] Stoichiometry of the Interaction of Refolded TFPI with FactorX_(a)

[0132] Inhibition of bovine factor X_(a) by the active refolded E. coliTFPI was examined by measuring the residual amidolytic activity usingSpectrozyme X_(a). The molar ratio of TFPI to bovine factor X_(a) thatresulted in the complete inhibition of the latter was 1:1 (open circle).For comparison, the stoichiometry of interaction of SK Hepatoma TFPIwith bovine factor X_(a) was also 1:1 (closed circle).

[0133] Inhibition of Tissue Factor-Induced Coagulation

[0134] The ability of the active, refolded E. coli TFPI to inhibittissue factor-induced coagulation in human plasma was compared with thatof the purified SK Hepatoma TFPI. The activity of the E. coli TFPI wasapproximately two fold more active than SK Hepatoma TFPI on a per molbasis as judged from the concentrations of each TFPI that produce thesame prolongation of clotting time. TABLE 1 Summary of refold andpurification of active E. coli TFPI. Specific Volume Total activity^(a)A₂₈₀ nm (ml) A₂₈₀ nm (Sk unit/mA) Yield Starting — — — — — material 0.56g inclusion body Sulfonated 6.1 25 153 0 — inclusion body HiLoad Q pool0.8 46 37 0 — Refold mixture 0.035 1050 37 0.66 100 Mono S pool 0.142 486.8 2.0 18

Example 2

[0135] This example illustrates the effectiveness of using LACI to treatpatients susceptible to or afflicted with sepsis. In particular, thisexample illustrates the effectiveness of using LACI to treat asepsis-associated coagulation disorder, namely, DIC.

[0136] Recombinant TFPI was expressed as a glycosylated protein usingmouse C127 cells as host and was purified by chromatography on amonoclonal antibody convalently attached to Sepharose 4B as described byDay et al. [Blood 76, 1538(1990)].

[0137] Baboons, 9 month of age weighing 9-13.9 kg, were randomlyselected for LACI or excipient pretreatment (1 hr or 15 min) protocol.Each baboon is immobilized with ketamine hydrochloride, 14 mg/kgintramuscularly on the morning of the study and slowly anesthetized withsodium pentobarbital (˜9 mg/kg) via a percutaneous catheter positionedin the cephalic vein and brachial vein. The femoral artery and onefemoral vein are cannulated aseptically to measure aortic pressure,obtain blood samples, and for infusion of LACI, live organisms, isotonicsodium chloride and sodium pentobarbital. Animals were pretreated withLACI [3.5 mg LACI per ml of excipient (150 mM sodium chloride and 20 mMsodium sulfate)] or excipient control as an I.V. bolus 40 μg/kg over 15minutes and then as an infusion at 5.6 μg/kg/min for 545 minutes in theleft cephalic vein. Baboons were challenged at time 0 with either 3ml/kg (4×10¹⁰) or 4 ml/kg (5×10¹⁰) of live E. coli. The actual dosingschedule and group assignment appear below: Time of Test Article # ofAnimals Administration Average Bacterial Group Males Females (min.) Dose(cfu/kg) 1 1 4 Excipient 4.1 × 10¹⁰ −60(3) −15(2) 2 1 2 LACI 3.8 × 10¹⁰−60(3) 3 0 2 Excipient 5.4 × 10¹⁰ −15(2) 4 0 3 LACI 4.9 × 10¹⁰ −15(3)

[0138] TABLE 2 Individual Animal Fibrinogen Level (% of Time Zero) +240+360 +720 −60/−15 0 +60 +120 (min.) (min.) (min.) Group 1 100 100 100 6740 23 16 100 100 93 85 16 1 1 100 95 100 68 32 25 15 100 100 100 100 298 6 100 100 85 64 52 24 20 Average 100.0 99.0 95.6 76.8 33.8 16.2 11.6STD DEV 0.0 2.0 6.0 13.7 11.9 9.8 7.0 Group 2 100 84 84 64 84 84 84 100100 86 100 86 108 86 100 83 108 108 100 95 108 Average 100.0 89.0 92.790.7 90.0 95.7 92.7 STD DEV 0.0 7.8 10.9 19.1 7.1 9.8 10.9 Group 3 100100 100 100 44 15 8 100 100 100 82 19 7 6 Average 100.0 100.0 100.0 91.031.5 11.0 7.0 STD DEV 0.0 0.0 0.0 9.0 12.5 4.0 1.0 Group 4 100 100 121121 100 100 83 100 90 90 85 79 62 60 100 80 91 80 72 75 64 Average 100.090.0 100.7 95.3 83.7 79.0 69.0 STD DEV 0.0 8.2 14.4 18.3 11.9 15.8 10.0

[0139] TABLE 3 Individual Animal Fibrin Degradation Products (μg/ml)+240 +720 −60/−15 (min.) (min.) Group 1 10.00 320.00 320.00 10.00 80.00320.00 10.00 80.00 160.00 10.00 80.00 160.00 10.00 10.00 160.00 Average10.00 114.00 224.00 STD DEV 0.00 106.51 78.38 Group 2 10.00 10.00 10.0010.00 10.00 10.00 10.00 10.00 10.00 Average 10.00 10.00 10.00 STD DEV0.00 0.00 0.00 Group 3 10.00 40.00 160.00 10.00 20.00 160.00 Average10.00 30.00 160.00 STD DEV 0.00 10.00 0.00 Group 4 10.00 10.00 20.0010.00 10.00 80.00 10.00 20.00 40.00 Average 10.00 13.33 46.67 STD DEV0.00 4.71 24.94

[0140] There was a clear effect by LACI on fibrinogen levels in the E.coli treated animals. A drop in fibrinogen is prominent in the excipientcontrols (Groups 1 and 3) from 240 minutes (i.e., two hours after theend of bacterial infusion) and on. The drop was substantially preventedby LACI pretreatment when the baboons were challenged with lower dosebacteria (Group 2), and attenuated when the animals are challenged withthe higher dose bacteria (Group 4).

[0141] The generation of fibrin degradation products was not detectablein Group 2, and slowed down and reduced in Group 4 animals as a resultof pretreatment with LACI. The differences in the above coagulationparameters among the groups are not as prominent at 720 minutes possiblydue to the fact that the LACI infusion was stopped at 540 minutes andthat a certain circulating level of LACI may be necessary to maintain aneffect.

[0142] In addition to the above analyses, histopathology studies whereintissues of all groups of the above baboons were processed, stained withhematoxylin and cosin, and examined by light microscopy. The kidneys,lungs, adrenals, liver and spleen appeared to be the main organsaffected by the E. coli challenge. Reduced pathology in some targetorgans such as adrenals and kidneys was observed.

[0143] Thus, the conclusion drawn from the above is that the effect ofLACI on septic shock is evident, particularly in view of the attenuationof the fibrinogen drop end generation of fibrin degradation products,and the reduced pathology in some target organs, such as the adrenal andkidney.

Example 3

[0144] This example illustrates the effectiveness of using LACI topromote survival in patients which are susceptible to or afflicted withsepsis. In particular, this example illustrates the effectiveness ofusing LACI to treat gram-negative sepsis. LACI was prepared by themethod described above in Example 1.

[0145] Male and female Papio anubis baboons (7.6±2.4 kg) from theCharles River Primate Center (Wilmington, Mass.) were quarantined for aminimum of thirty days in the University of Oklahoma Animal Facility(Oklahoma City, Okla.).

[0146] Each baboon was immobilized with ketamine hydrochloride, 14 mg/kgintramuscularly on the morning of the study and slowly anesthetized withsodium pentobarbital (−9 mg/kg) via a percutaneous catheter positionedin the cephalic vein. To compensate for insensible fluid loss, theanimals were infused with isotonic saline at a rate of 3.3 ml/kg/hr for12 hours via the barachial vein in the right leg. LACI or PBS buffercontrol was administered to the animals through the brachial vein 30minutes after the administration of bacteria. LACI was administered at aloading dose over fifteen minutes and simultaneously started acontinuous infusion of LACI for an additional 675 minutes (counting fromstart of bacterial infusion, which was defined as time zero).

[0147]E. coli 086: K61H were used to inoculate tryptic soy broth agar(2); viability counts of the inoculum were determined by standarddilution techniques. At time zero, baboons received an infusion of4.5×10¹⁰ live bacteria per kg body weight (4 mls/kg), administeredthrough a percutaneous catheter in the right cephalic vein by continuousinfusion for 2 hours.

[0148] The femoral artery and one femoral vein were cannulatedaseptically to measure mean systemic arterial pressure, obtain bloodsamples and for antibiotic administaration. Gentamicin was given (9mg/kg i.v.) at end of E. coli infusion, i.e., at T+120 min. for 30minutes and then 4.5 mg/kg at T+300 min. and T+540 minutes for 30 min.Gentamicin (4.5 mg/kg IM) was then given at the end of the experimentand once daily for 3 days.

[0149] Animals were maintained under anaesthesia and monitoredcontinuously for 12 hours. Blood samples were collected hourly forhematology, clinical chemistry, cytokines (TNF, IL-6) and LACIdeterminations. Similarly, respiration rate, heart rate, mean systemicarterial pressure and temperature were monitored hourly.

[0150] Animals surviving 7 days were considered survivors andsubsequently euthanized for necropsy at the 8th day.

[0151] See Hinshaw, L. B., Archer, L. T., Beller-Todd, B. K., Coalson,J. J., Flournoy, D. J., Passey, R., Benjamin, B., White, G. L. Survivalof primates in LD₁₀₀ septic shock following steriod/antibiotic therapy,J. Surg. Res., 26, 151-170 (1989), and Hinshaw, L. B., Brackett, D. J.,Archer, L. T., Beller, B. K., Wilson, M. F. Detection of the“hyperdynamic state” of sepsis in the baboon during lethal E. coliinfusion, J. Trauma, 23, 361-365, (1982); which are incorporated hereinby reference.

[0152] Results of this study are shown in Table 4. TABLE 4 E. coil LACIBaboon Data Fibrinogen Platelet Ct. Blood Pressure Baboon 240′ 720′ 702′Hemolysis 3 hr 12 hr Recovery at 24 hr. Survival # % of T = O % of = O 6hr 12 hr % of T = O Consciousness Alertness Mobility Time Controls 3 <1<1 27 −− −− 61 41 18 hrs 6 8 <1 21 − − 51 84 −− −− −− 33{fraction (1/2)} hrs 18 4 <1 19 − − 65 84 − − − 66 hrs 19 <1 <1 22 −− −− 89 122 − − −7 days 20 4 <1 26 + + 53 45 − − − 83 hrs Low Dose (Loading Dose 0.7mg/kg: Maintenance Dose 3.0 μg/kg/min) 7 78 8 37.5 − −− 62 50 18 hrs 855 50 32.2 − − 38 69 ++ ++ ++ 53 hrs 10 32 10 15 − − 73 63 + + − 7 days11 93 83 26 + + 63 89 ++ ++ − 7 days 16 43 43 33 − − 75 80 − − − 7 days17 74 84 30 + + 75 90 ++ + + 7 days High Dose (Loading Dose 1.0 mg/kg:Maintenance Dose 9.5 μg/kg/min) 4 77 22 70 + + 56 87 + + + 7 days 5 79113 59 + + 75 77 ++ ++ ++ 7 days 12 116 71 48 + + 79 68 ++ ++ ++ 59.5hrs 13 86 49 30 + + 61 82 ++ ++ ++ 7 days 14 88 105 30 + + 74 71 ++ ++++ 7 days 15 54 54 41 + + 78 90 ++ ++ ++ 7 days

Example 4

[0153] Production of LACI

[0154] A. Aged Cells

[0155] Human umbilical vein endothelial cells (HuVec) were plated andmaintained in a standard tissue culture medium. They were aged for 32-36days, fed twice a week with fresh medium, and the medium supernatant wasremoved after 32 days (called conditioned medium or CM). The CMcontained LACI.

[0156] B. Induced Cells

[0157] The same HuVec cells were plated and maintained in a tissueculture medium for 24-48 hours and then they were contacted with variousconcentrations of tumor necrosis factor (TNF) for 34 days. The mediumcontaining LACI was removed and is called TNF CM.

Example 5 LACI Inhibition of Sepsis

[0158] The following assay was devised to measure the inhibition ofsepsis by LACI. HuVec cells were plated and incubated for 48 hours.Bacterial lipopolysaccharide (LPS) was added as an inducer of sepsis.The addition of LPS was the best way to stimulate a sepsis-like responsewhich was broader than simple coagulation. When the inducer was added, atest sample was added to examine its effect on the LPS effect on theendothelial cells. The sample that was tested contained LACI. The cellswere incubated between 4 and 5 hours and then chromozyme was added. Thechromozyme contains Factors II, VII, IX, and X. This first methodmeasured the inhibition of tissue factor induction and inhibition ofactivity. In an alternative of the present assay, which measuresinhibition of tissue factor activity, the sample was added together withthe chromozyme and then incubated for 45 minutes. LACI inhibitoryactivity was measured by reading optical density (due to color changes)in a spectrophotometer at A₄₀₅.

[0159] Aged and TNF induced condition medium was prepared as in Example4. FIG. 2 displays a dose dependent inhibition of tissue factor activityby a substance contained in the respective media shown in the figure.The nature of the substance was identified by the following experimentwhich involved inhibition of tissue factor activity determined asfollows: HuVec cells were prepared for the assay. One cell sample wasleft untreated as a control. Another cell sample was induced with LPSwithout the addition of any potential inhibitor. Subsequently, sixclasses of samples were run using aged and TNF condition mediumcontaining LACI with 0, 10, and 100 mg of LACI antibody. FIG. 3 showsthe result of this experiment. For example, (Lane 1 starting from theleft) was the control and very little tissue factor activity wasdetected. Lane 2 shows 100% of tissue factor activity and induction byaddition of LPS. Lanes 3, 4, and 5 show linearly increasing amounts ofactivity (and thus induction) depending on the amount of anti-LACIantibody. For example, the 0 concentration (Lane 3) showed that verylittle tissue factor activity was detectable, suggesting lack of tissuefactor induction. This indicated that LACI inhibited the activity of thetissue factor induced by LPS. Lanes 4 and 5 show a similar result,however, the amount of tissue factor activity/induction increased aslarger amounts of LACI were neutralized by the anti-LACI antibody. Lanes6, 7, and 8 (with TNF conditioned medium) also display a nearlyidentical magnitude of inhibition of tissue factor activity as thatshown for Lanes 3, 4, and 5. To confirm the identity of the substance inthe conditioned media, we used various concentrations of highly purifiedLACI in the absence or presence of neutralizing antibodies. The resultsmatch the findings utilizing aged and TNF induced conditioned media. SeeFIG. 4.

[0160] These data indicate that LACI will inhibit the effects of LPS onHuVec cells in a concentration dependent manner and this effect may bereversed upon the addition of various concentrations of neutralizingantibodies to LACI. Furthermore, this model proves that LACI can be usedto treat sepsis, and its effects were not simply restricted to itsanticoagulant properties.

Example 6 Treatment of Human Patients Using LACI

[0161] Human patients which are affected by sepsis may betherapeutically treated by using LACI. When the patient presentsthemselves with increased temperature, drop in blood pressure, adecrease in white cell count, and a drop≧20% in fibrinogen, LACI isadministered intravenously as a bolus dose of 3-10 mg/kg and as aninfusion of 10-20 μg/kg/min for 34 hours. Alternatively, LACI may beadministered at a continuous rate of approximately 10 mg/kg/min for 3days or for 4 hours daily for 34 days. Antimicrobial therapy or broadspectrum antibiotics are administered to the patient along with theLACI.

[0162] LACI is given prophylactically in the same manner.

Example 7

[0163] In this experiment, highly purified recombinant LACI (6 mg/kg)was administered either thirty minutes or four hours after the start ofa lethal intravenous E. coli infusion in baboons. Early post treatmentof LACI resulted in a) permanent 7 day survivors (5/5) with significantimprovement in quality of life, while the mean survival time for thecontrols (5/5) was 39.9 hrs. (no survivors); b) significant attenuationsof the coagulation response and various measures of cell injury, withsignificant reductions in pathology observed in E. coli sepsis targetorgans including kidneys, adrenals and lungs. LACI administration didnot affect the drop in mean systemic arterial pressure, the increases inrespiration and heart rate or temperature changes associated with thebacterial infusion. LACI treated E. coli infected baboons had twentyfold lower IL-6 levels than their phosphate buffered saline treatedcontrols. In contrast to the earlier 30 minute treatment, theadministration of LACI at four hours i.e., 240 minutes, after the startof bacterial infusion resulted in prolongation of survival time, withforty percent improvement in survival rate (two survivors) and someattenuation of the coagulopathic response, especially in animals inwhich fibrinogen levels were above 10% of normal at the time of LACIadministration.

Recombinant Tissue Factor Pathway Inhibitor

[0164] LACI was expressed in the human hepatoma cell line SK Hep asdescribed in Wun et al. 1992, Thrombosis Haemost., 68:54-59. Detectionof Bacterial Endotoxins with the Limulus Amebocyte Lysate Test, Alan R.Liss, Inc., NY. The material was purified by standard techniques toprovide >95% pure preparations. LACI was formulated in 150 mM NaCl and20 mM NaPO₄ (pH 7.2), which served as the excipient control. Finalprotein concentration in a LACI sample ranged from 2.3-3.7 mg/ml,determined by amino acid composition; endotoxin levels ranged from 8 to27 endotoxin units per 15 milligrams of protein. LACI lots weremonitored for biological activity using a tissue factor inhibition assay(Boze et al., Blood 71:335-343 (1988)).

[0165] Baboons

[0166] Male and female Papio anubis baboons (7.6±2.4 kg) from theCharles River Primate Center (Wilmington, Mass.) were quarantined for aminimum of thirty days in the University of Oklahoma Health SciencesCenter Animal Resource Facility (Oklahoma City, Okla.). Animals werefree of infections or parasites with hematocrits≧36%.

[0167] Bacteria

[0168] Escherichia coli 086:K61H organisms (ATCC 33985; Rockville, Md.)were isolated from a stool specimen at Children's Memorial Hospital,Oklahoma City. They were stored in the lyophilized state at 4° C. aftergrowth in tryptic soybean agar and reconstituted and characterized asdescribed in Hinshaw et al., J. Trauma 23:361-365 (1982).

[0169] Assays

[0170] Endotoxin Measurement

[0171] Endotoxin levels in LACI preparations and the excipient bufferwere monitored by the limulus amebocyte lysate test (Wun et al., Thromb.Haemost. 68:54-59 (1992)). LPS from E. coli (B5505; Mallinckrodt, St.Louis, Mo.) were included as a standard. The detection limit of theassay was 10 endotoxin units (E.U.)/ml.

[0172] TNF ELISA

[0173] Baboon TNF levels in plasma were measured using an ELISAdeveloped for detecting human TNF (Creasey et al., Circ. Shock 33:84-91(1991)): a purified monoclonal anti-TNF antibody (24510E11) was bound tomicrotiter plate wells (Dynatech Immunolon I, Fisher). Unoccupiedbinding sites on the plastic were then blocked with bovine serum albumin(BSA). Aliquots of standard concentrations of purified recombinant humanTNF or baboon plasma samples were incubated in duplicate. ELISA wellswere exposed to horseradish peroxidase (HRP)-conjugated affinity packedpolyclonal rabbit antibody to recombinant human TNF followed by0-phenylenediamine substrate as chromogen. Wells were rinsed repeatedlywith phosphate-buffered saline solution (PBS, Ph 7.5) between successiveincubations. Optical density (OD) was read on an automateddual-wavelength plate reader at 490 nm (Bio-Tek Instruments). Thedetection limit for baboon TNF in this assay was 0.5 ng/ml.

[0174] IL-6 Bioassay

[0175] IL-6 bioactivity was quantified in baboon plasma using theIL-6-dependent murine hybridoma cell line B9, using IL-6 commerciallyavailable from Amgen, Inc. (Thousand Oaks, Calif.), as the assaystandard (Creasey et al., supra). The detection limits of this assaywere 10 pg/ml.

[0176] LACI Levels

[0177] A competitive fluorescent immunoassay for LACI was used aspreviously described in Novotny et al., Blood 78:394400 (1991): a rabbitanti-LACI IgG was used to capture LACI in the sample to be tested andFITC-LACI (HepG2) was added to quantitate the number of anti-LACIbinding sites remaining. Standard curves were constructed usingdilutions of pooled human plasma (George King Biomedical, Overland Park,Kans.) or of pure HepG2 LACI.

[0178] The LACI functional assay (tissue factor-inhibition assay) is athree-stage clotting assay. Briefly, in the first stage, the sample tobe tested is incubated with crude brain tissue factor, factor X, factorVII, and calcium. After 30 minutes of incubation, additional factor X isadded and 1 minute later factor X-deficient plasma is added and time toclot is measured in a fibrometer. Residual factor VII(a)/tissue factoractivity in the second stage of the assay is inversely proportional tothe LACI concentration in the test sample. Thus, prolongation of theclotting time reflects higher LACI activity. Standard curves wereconstructed using dilutions of pure HepG2 LACI.

[0179] Pharmacokinetic Analysis

[0180] The data for each baboon (μg LACI/ml plasma at various sampletimes) were fit to a two-compartment model. The model parameters weredetermined by nonlinear least squares curve-fitting procedures using thePKDAAS data analysis system (developed for the VAX computer at ChironCorporation deposited at the U.S. Copyright Office as registration No.TXU 416-977). Corrected concentrations at each time, C(t), were weightedas the reciprocals of each concentration squared. The weighted valueswere then fitted to individual subjects' curves using the followingbiexponential equation:

C(t)=(DOSE/VC)*[(1−B)*2^(−t/α) +B*2^(−t/β)],

[0181] where t is time and VC, B, α, and β are model parameters. The sumof the coefficients was normalied to 1.0. The systemic clearance (CL)was then calculated from:

CL=VC/MRT, where

MRT=[(1−B)*α+B*β]ln(2).

[0182] Statistical Analysis

[0183] Data were analyzed with the students' t-test to determinesignificant differences (p<0.05) in means between groups at given times.The analysis of variance (ANOVA) and the multicomparison Duncan's testwere used to determine significant differences between means at time 0and subsequent times within groups. The Fisher's exact test was used todetermine significant differences between groups with respect tosurvival rates.

[0184] Pharmacokinetic Studies

[0185] To establish the appropriate LACI dosage for the E. coli septicshock model, we performed a pharmacokinetic study in three healthybaboons. FIG. 5 shows that administered as a bolus at 0.5 mg/kg, LACIexhibited a two phase half life; an alpha phase of approximately twominutes and a beta phase of about two hours. These data were thenmodeled as described above to identify the necessary LACI dosage toachieve a circulating LACI serum concentration of 2 μg/ml, which wasarbitrarily defined as the desired LACI blood concentration since it hasbeen reported that endogenous levels of LACI in primates isapproximately 0.1 μg/ml (Novotny et al., J. Biol. Chem. 264:18832-18837(1989)). Thus to achieve a 20-fold increase in LACI serum concentrationsin the baboons, we administered LACI at a loading dose of 700 μg/kg anda maintenance dose of 10 μg/kg/min (i.e. a total dose of 6,000 μg/kg)started simultaneously, 30 minutes after the start of the E. coliinfusion.

[0186] Experimental and Infusion Procedures

[0187] Each baboon was immobilized with ketamine hydrochloride, 14 mg/kgintramuscularly on the morning of the study and slowly anesthetized withsodium pentobarbital (˜9 mg/kg) via a percutaneous catheter positionedin the cephalic vein as described in Hinshaw et al., J. Surg. Res.28:151-170 (1989). To compensate for insensible fluid loss, animals wereinfused with isotonic saline at 3.3 ml/kg/hr for 12 hours via-thebrachial vein 30 minutes or 240 minutes, respectively, after theadministration of bacteria. LACI was administered at a loading dose of700 μg/kg for 15 minutes and a continuous infusion of LACI at 10μg/kg/min was given for an additional 525 minutes (counting from startof bacterial infusion, which was defined as time zero). To deliver thesame total LACI dose per baboon, animals treated at +240 minutesreceived a loading dose of 2.8 μg/kg for fifteen minutes andsimultaneously received a continuous infusion of LACI at 10 μg/kg/minfor 480 min.

[0188]E. coli 086:K61H were used to inoculate tryptic soy broth agar,and viability counts of the inoculum were determined by standarddilution techniques. At time zero, baboons received an infusion of≧4.5×10¹⁰ live bacteria per kg body weight (4 ml/kg), administeredthrough a percutaneous catheter in the right cephalic vein by continuousinfusion for 2 hours.

[0189] The femoral artery and one femoral vein were cannulatedaseptically to measure mean systemic arterial pressure, obtain bloodsamples and for antibiotic administration. Gentamicin was given (9 mg/kgi.v.) at the end of E. coli infusion, i.e., at T+120 for 30 minutes andthen 4.5 mg/kg at T+360 and T+540 minutes for 30 min. Gentamicin (4.5mg/kg IM) was then given at the end of the experiment and once daily for3 days.

[0190] Animals were maintained under anesthesia and monitoredcontinuously for 12 hours. Blood samples were collected hourly forhematology, clinical chemistry, cytokines (TNF, IL-6), and LACIdeterminations. Similarly, respiration rate, heart rate, mean systemicarterial pressure and temperature were monitored hourly. Animals werecontinuously observed for the first 30 hours of the experiment. Thosesurviving 7 days were considered permanent survivors and weresubsequently euthanized with sodium pentobarbital for necropsy at the8th day.

[0191] Ten baboons (5 LACI treated and 5 excipient controls) wereintravenously administered 2 hour lethal infusions of E. coli. Table 5shows that LACI rescued five of five E. coli treated baboons who becamepermanent survivors. The mean E. coli dosage of the LACI treated was5.7×10¹⁰ CFU/kg and all animals survived more than 7 days. The mean E.coli dosage of the excipient control group was 5.5×10¹⁰ CFU/kg and themean survival was 39.9 hours (Table 5). The mean weight of the excipientcontrol group was 8.4 kg (range 5.9 to 12.1 kg) and that of the LACItreated was 6.8 kg (range 5.2 to 8.0 kg). Two females and three malescomposed the excipient control group, while the LACI treated groupconsisted of five males. There was no difference in the mean dose of E.coli administered to each group (p>0.05) nor in the animals' weights(p>0.05).

[0192] LACI treated baboons moved about the cage energetically, consumedsome food and drank water normally within 24 hours of receiving lethalE. coli (LD₁₀₀). The excipient control baboons, however, were verylethargic, appeared to have difficulty breathing and exhibited multiplepetechiae over their bodies indicating the occurrence of DIC in thedermal microvasculature.

[0193] Coagulation and Hematological Responses to LACI Administration at+30 Minutes

[0194] To determine the mechanism by which LACI protected thebacterially infected baboons we measured selected physiologic parametersassociated with coagulation, clinical chemistries and the inflammatoryresponse. FIG. 6 shows that many of the coagulopathies associated withthe bacterial infection were inhibited and/or attenuated in the LACItreated baboons. Fibrinogen levels in excipient control animals droppedby approximately 80% by 3 hours, while the LACI treated baboonsexperienced only a 20% drop (p<0.0001). Similarly, the rise in fibrindegradation products at 240 and 720 minutes, as a marker of fibrinogenconsumption, was not evident in the LACI treated animals as compared tothe controls (p<0.05).

[0195] Activated partial thromboplastin time (APTT) and prothrombin time(PT) were extremely prolonged at times beyond four hours in theexcipient controls (FIG. 6). APTT increased from 37 to 208 and then to226 seconds while PT increased from 14 to 58 and then to 137 seconds, atfour and 12 hours, respectively. In contrast, APTT increased from 32 to45 to 60 seconds at four and 12 hours, respectively, and PI increasedfrom 15 to 18 seconds to 22 seconds at four and 12 hours, respectively,in the LACI treated baboons (p<0.05).

[0196] A gradual drop in platelet cell concentration was noted in theexcipient controls and in the LACI treated baboons over the 12 hourobservation period (FIG. 6). LACI treatment, however, retards the dropand is most apparent at ≧4 hours. The mean platelet concentration of thecontrol group at four, six and twelve hours were 102.8±26, 69±20 and43±5.0. In contrast, the mean platelet concentration of the LACI treatedgroup at the same times were 249±44, 236±35, and 153±31, respectively.

[0197] Despite the lack of visible hemolysis in the LACI treated plasmasamples, the hematocrit decreased with time and was lower at 12 hours inthe experimental (treated) group, 36±2%, as compared to the controlgroup, 44±2.% (p<0.05). Furthermore, the mean 7 day hematocrit value ofthe survivors was also low as compared to baseline: 28±1% versus 42±0%.

[0198] Consistent with the hematocrit results the red blood cellconcentration dropped only slightly over the initial 12 hours in boththe control (4.94±0.21 to 4.4±0.11) and LACI treated groups (5.20±0.10to 4.88±0.17), and a low (3.42±0.2×10⁶) red cell concentration wasobserved in the survivors.

[0199] Leukopenia occurred to the same degree in the LACI treated andcontrol group, the lowest values (˜1.48×10³/μl) recorded at 2 hours;however, the white blood cell concentration was found elevated at 7 daysin the survivors with a mean of 19.2±3.5 as compared to the base line of9.0±1.5×10³/μl.

[0200] Clinical Responses to LACI Administration at +30 Minutes

[0201] Respiration and heart rate increased in both groups. Respirationrate rose quickly after the start of the bacterial infusion and remainedelevated for the 12 hour period. Similarly, heart rate increaseddramatically, from 120 beats/min to 200 beats/min, within the first twohours of E. coli infusion and remained elevated during the 12 hours.

[0202] Mean systolic arterial pressure (MSAP) and temperature equallydeclined in the LACI treated and control groups. A dramatic decrease inMSAP was observed at the end of the bacterial infusion. MSAP declinedfrom 107+5 mm Hg to 69±5 at two hours and then gradually returned to93±11 by 10 to 12 hours in the control group. Similarly, MSAP declinedfrom 115±9 to 74±3 at 2 hours and rose to 85±7 from 6 hours on to 12hours. The ten baboons had a decreased temperature response to the E.coli infusion. The mean excipient control temperature at the start ofthe experiment was 37.3±0.1° C. and declined slowly to 34.7±2.2° C. at12 hours. The mean LACI treated temperature was initially 37.0±0.3° C.and changed minimally over the 12 hours where it was 36.9±0.2° C.

[0203] Blood Chemistries

[0204] Table 6 summarizes clinical chemistries of the E. coli infectedand treated ten baboons. Increases in serum creatinine, total bilirubin,uric acid, lactic acid, triglycerides, anion gap, chloride and sodiumwere measured at 12 hours. The magnitude of the increases, however, waslower in the LACI treated animals than the excipient controls (p<0.05).Changes in the concentrations of the following parameters were observed:albumin, alkaline phosphatase, AST, BUN, calcium, cholesterol, CK,carbon dioxide, cortisol, potassium, lactic dehydrogenase, phosphorous,SGPT and total protein. Their increases or decreases in concentrationwere not affected by the LACI treatment (p>0.05). However, the meanconcentrations of albumin, urea nitrogen (BUN) and lactate did notreturn to baseline values in the LACI treated animals (i.e. thesurvivors) at 7 days. Specifically, albumin concentrations were 2.7±0.2at 7 days as compared to 3.7±0.1 at the start of the experiments. Thusalbumin was reduced by about 25%. Similarly, serum values of ureanitrogen (BUN) at 7 days was 13.8±2.1 versus 29.6±3.9 at the beginningof the experiment. Finally, lactate concentrations were increased byabout 3-fold in the survivors. The mean baseline lactate concentrationsof these animals was 1.7±0.5 meq/L at the start of the procedure andincreased to 5.7±1.2 meq/L at 7 days.

[0205] Increases in glucose concentration were observed within two hoursin both groups (p<0.05). Mean values fell gradually beyond the initialincrease but remained consistently higher in the LACI treated animals(p<0.05) until 12 hours. Increases in arterial pH occurred in bothgroups.

[0206] TNF and IL-6 Levels

[0207] Plasma TNF concentrations were elevated in both the excipientgroup and LACI treated baboons. Consistent with our previous studies(Creasey et al., Circ. Shock (1991) 33:84-91), peak TNF levels were at120 min, i.e. at the end of E. coli infusion. LACI treatment did notappear to affect the rise in serum TNF concentrations nor the kineticsof its release (Table 7). Plasma IL-6 concentrations also increased withtime in the excipient control group, where IL-6 levels started at 26-39picograms and rose to 100-120 nanograms beyond four hours (Table 8).Interestingly, plasma IL-6 concentrations in the LACI treated animalswere lower than those of the control group, especially at and beyondfour hours. IL-6 concentrations were about 20-fold lower in the LACItreated than the excipient controls at 12 hours (p<0.05).

[0208] Administration of LACI at +240 Minutes

[0209] To determine the time beyond which LACI may no longer beeffective in attenuating the E. coli shock, we delayed theadministration of LACI to two hours after the end of the bacterialinfusion. Fibrinogen consumption and the generation of fibrindegradation products were to be clearly evident at four hours. Table 8shows that the mean E. coli dosage of the excipient control group inthis series of experiments was 5.68 (±2.6)×10¹⁰ CFU/kg and the meansurvival time of 28.2±9.6 hours. The mean E. coli dosage of the LACIgroup was 5.43 (±0.19)×10¹⁰ CFU/kg and the mean survival time of 99±29hours. Two of the five LACI treated animals were 7 day survivors(p<0.05). There was no difference in the mean weight or E. coli dosageadministered to each of the above groups (p>0.05).

[0210] Biological and Biochemical Effects of Administration of LACI at+240 Minutes

[0211] The administration of LACI two hours after the end of the twohour bacterial infusion was effective in slightly attenuating thecoagulopathic response as evident by decreases in FDP levels, andprothrombin time at >12 hours. Consistent with +30 minutes, IL-6 levelswere two-fold lower in the LACI treated baboons than their excipientcounterparts at 12 hours. No significant differences in fibrinogenconcentrations, APTT and platelet cell concentration were noted at 12hours between the excipient control and the LACI treated baboons.However, fibrinogen levels at day 7 in the two animals that survivedwere slightly elevated; FDP, APTT and PT values were back close tonormal while platelet cell concentrations were normal in one (435) andlower in the other (97).

[0212] Although the red blood cell count and hematocrit fluctuatedslightly over time in both groups during the first 12 hours, the twosurvivors had lower hematocrits at day 7 (35 and 19%) as compared to thestart of the procedure (43 and 41%). Similarly, red blood cellconcentration was 4.0 and 2.7×10⁶/mm³ on day 7 versus 4.7 and4.5×10⁶/mm³ at day 0.

[0213] Clinical chemistries were measured for the ten baboons comprisingthe plus four hour study as we had performed for the plus thirty minutestudy. We observed minimal differences between the excipient control andLACI treated baboons at twelve hours. However, consistent with the plus30 minute study, lactate levels were higher in the LACI treated than thecontrols (p<0.05) at 12 hours and remained elevated in the two thatsurvived 7 days (13.2 and 4.0 mg/dl versus 0.5 and 0.6 mg/dl at time 0).In contrast, uric acid levels were slightly lower in the LACI treatedgroup than the controls at 12 hours and returned to normal levels in thetwo LACI treated survivors.

[0214] Similar to the plus 30 minute study, all the animals treated at240 minutes experienced leukopenia, and a gradual but small rise in WBCcount over the twelve hours. Furthermore, the two 7 day surviving LACItreated baboons had elevated WBC counts (12.5 and 21.8×10³ cells/mm³) atday 7 as compared to 5.1 and 8.0×10³ cells/mm³ at time zero; this trendis similar to that observed in the survivors of the baboons treated at+30 minutes with LACI.

[0215] Pathological Results

[0216] Post-mortem examinations were conducted on all baboons.Surveillance of animals was continuous for the first 36 hours;consequently tissues were removed for analysis within minutes afterdeath thereby avoiding post-mortem autolytic changes. Lungs, liver,adrenals, kidneys, spleen, and gall bladder were target organs of the E.coli bacterial infusion. Specifically, animals that received excipient+E. coli suffered from severe congestion, hemorrhage, fibrin deposition,edema and massive accumulation of leukocytes in the lungs and liver,severe congestion of medullary sinusoids in the spleen and significantevidence of tubular necrosis and thrombosis within the kidneys andsevere cortical congestion in the adrenals. Organs not affected by E.coli were stomach, heart, pancreas and small and large intestines. LACIprotected the liver, adrenals, kidneys, spleen and gall bladder in whichonly mild to no pathology were observed. The degree of protection wasslightly diminished in the lungs, in which moderate vascular congestion,and mild leukocyte accumulation were observed.

[0217] Results from the present study demonstrated that LACI rescued onehundred percent of the baboons given LD₁₀₀ doses of E. coli whenadministered thirty minutes after the start of the bacterial infusionwhen more than 1×10¹⁰ organisms/kg had already been introduced into theblood of the baboons. In addition, LACI rescued forty percent of thebaboons when given two hours after the end of the bacterial infusioni.e. when greater than 5×10¹⁰ organisms/kg had been infused and many ofthe baboons' host defense mechanisms had been triggered for two hours.

[0218] TNF levels peaked at the end of the E. coli infusion i.e. at twohours, while IL-1β and IL-6 levels started to appear (Creasey et al.,Circ. Shock 33:84-91 (1991)); the decline and consumption of fibrinogenand generation of fibrin degradation products become more easilydetectable between three and four hours (De Boer, J. P. et al., Circ.Shock (In press 1992)). This study shows that LACI could prevent, slowdown and even reverse the consumption of fibrinogen, when administeredas late as four hours after the start of a lethal bacterial infusion.

[0219] In addition to attenuating coagulation, LACI attenuated thedegree of cell injury (creatinine, uric acid, lactic acid) and metabolicacidosis (anion gap, chloride and sodium) so clearly evident in thecontrols. Consistent with the decreased serum levels of many of thesemarkers of hypoxia, acidosis and cell injury, LACI afforded remarkablemorphological protection to kidneys, adrenals, liver, spleen and thelungs from pathological changes. The efficacy of LACI in baboonschallenged with lethal E. coli shows gram-negative shock is an acuteinflammatory disease of the vascular endothelium and that significantbenefit is achieved by transiently protecting the endothelium frominsults associated with gram-negative bacteria.

[0220] Previous studies have shown that within the first 30 minutes ofthe bacterial infusion, the PMN leukocyte concentration in circulatingblood fell sharply (Taylor et al., Colloquium Mosbach Molecular Aspectsof Inflammation (1991) Springer Verlag, Berlin Heidelberg, pp. 277-288),thrombin-antithrombin (TAT) complexes, tissue plasminogenactivator/plasminogen activator inhibitor (t-PA/PAI) and plasmin antiplasmin (PAP) complexes had started to appear (De Boer, J. P. et al.,Circ. Shock (1992) In press), and the activation of the complementcascade in lethal E. coli challenge was clearly evident (De Boer, J. P.et al., submitted). LACI treatment resulted in the prevention of tubularnecrosis and glomerular thrombosis in the kidneys; cortical congestion,hemorrhage, necrosis and leukocyte accumulation in the adrenals;prevention of vascular congestion and accumulation of leukocytes in theliver; prevention of medullary congestion, hemorrhage and necrosis inthe spleen; and fibrin thrombi deposition and edema formation in thelungs. LACI significantly attenuated leukocyte influx and vascularcongestion in the lungs. The two baboons that received LACI at fourhours and survived seven days showed a very similar prevention ofpathological changes as those described above. However, there was somemild edema and fibrin present in alveolar sacs of the lungs withmoderate leukocyte accumulation and vascular congestion. There was noevidence of multiple organ failure in any of the LACI treated baboonsthat survived seven days. This degree of protection is remarkable andunexpected given the delayed administration of LACI and the massivebacterial challenge afforded to the baboons.

[0221] The LACI-treated, E. coli challenged, 7 day survivorsdemonstrated a lower red blood cell concentration and an increase inleukocyte concentration. Histological examination did not reveal theoccurrence of hemorrhage in any tissue. Thus the lower hematocrit may beattributed either to hemodilution or to the slow generation oferythrocytes in the bone marrow. LACI toxicology studies with uninfectedbaboons may be necessary to resolve this matter.

[0222] The decreased IL-6 levels observed in the E. coli challenged andLACI treated baboons in the present study show was unexpected andsuggest that LACI either directly or indirectly exhibits an effect onthe inflammatory response. Thus, in addition to its anticoagulantactivity, a physiologic role of LACI is useful in the modulation of theinteraction of the coagulation pathway with various participants of theimmune system. TABLE 5 Weight, Sex, E. coli Dose and Survival Times ofControl and LACI* Treated Baboons at +30 min** Weight Mean Dose E. coliSurvival (kg) Sex (CFU/kg × 10¹⁰) Time (hrs) Control (E. coli +excipient control) 26 12.1 M 5.71 46 27 9.8 F 5.60 52.5 32 6.4 F 5.239.7 37 7.7 M 5.26 30.5 41 5.9 M 5.70 60.5 Mean 8.4 5.50 39.9 (±SE) ±1.1±0.11 ±9.0 Experimental (E. coli + LACI) 29 8.0 M 4.84 >168 30 7.5 M5.22 >168 31 7.3 M 6.05 >168 38 5.2 M 6.21 >168 40 6.1 M 6.15 >168 Mean6.8 5.69 168 (±SE) ±0.5 ±0.28 ±0.0

[0223] TABLE 6 Clinical Chemistry Summary of LACI Treated and ControlBaboons at +30 min** Control LACI (Mean ± STD error) Mean ± STD error)TO T + 12 hrs TO T + 12 hrs + 7 days p <0.05: Creat (mg/dL) 0.64 ± 0.052.68 ± 0.27 0.64 ± 0.09 0.92 ± 0.07 0.48 ± .07  T Bili (mg/dL) 0.16 ±0.02 1.35 ± 0.33 0.14 ± .02  0.30 ± 0.11 0.20 ± 0.05 Uric Acid (mg/dL)0.38 ± 0.07 0.93 ± 0.18 0.50 ± 0.0  0.50 ± 0.00 0.32 ± 0.07 Lactate(mEq/L) 0.94 ± 0.38 6.05 ± 0.59 1.74 ± 0.47 4.10 ± 0.34 5.70 ± 1.21Triglycerides 64 ± 7  283 ± 19  101 ± 25  161 ± 28  130 ± 42  (mg/dL)Anion GAP (mEq/L) 13.4 ± 0.75 19.25 ± 0.63  11.2 ± 1.24 11.25 ± .25 12.75 ± 1.03  Cl (mEq/L) 107.68 ± 3.73  109.58 ± 1.16  105.76 ± 0.52 117.62 ± 1.08  100.56 ± 6.61  Na (mEq/L) 150.58 ± 4.81  149.50 ± 0.65 146.04 ± 1.31  153.0 ± 1.0  142.26 ± 8.51  p >0.05 Alb (g/dL) 3.82 ±0.27 2.90 ± 0.33 3.66 ± 0.13  2.8 ± 0.06 2.68 ± 0.22 Alk phosp. (IU/L)827 ± 59  949 ± 62  933 ± 68  1032 ± 121  937 ± 117 AST (U/L) 40 ± 3 1531 ± 783  45 ± 5  710 ± 484 68 ± 8  BUN (mg/dL) 19.4 ± 1.9  39.0 ±4.1  29.6 ± 3.9  34.0 ± 3.2  13.8 ± 2.1  CA (mg/dL) 9.9 ± 0.4 7.1 ± 0.310.3 ± 0.2  7.9 ± 0.2 9.1 ± 0.5 Chol (mg/dL) 126 ± 10  94 ± 7  130 ± 3 86 ± 3  135 ± 16  CK (U/L) 604 ± 130 5979 ± 1705 795 ± 348 5594 ± 732 289 ± 79  CO₂ (mEq/L) 29.6 ± 2.0  20.4 ± 1.7  29.2 ± 0.9  23.7 ± 0.6 28.2 ± 1.9  Cortisol (μg/dL) 48.4 ± 7.9  120.2 ± 21.1  48.2 ± 12.2 110.5± 16.2  28.5 ± 2.6  K (mEq/L) 3.90 ± 0.14 4.75 ± 0.44 3.86 ± 0.14 4.32 ±0.10 3.62 ± 0.25 LDH (IU/L) 311 ± 46  3956 ± 1112 317 ± 40  1819 ± 621 433 ± 58  Phos (mg/dL) 5.94 ± 0.69 8.28 ± 0.59 4.78 ± 0.34 7.66 ± 0.673.96 ± 0.55 SGPT(IU/L) 69± 23 936± 507 49± 9  366± 281 101± 11  TotalProtein (g/dL) 7.00 ± 0.36 5.88 ± 0.35 6.62 ± 0.21 5.36 ± 0.18 5.92 ±0.36

[0224] TABLE 7 Individual Animal IL-6 Levels (ng/ml) LACI Administrationat +30 min TO +30 +120 +240 +360 +720 Control (E. coli + excipientcontrol) 26 .034 .027 21.5 102.3 347.2 468.5 27 .018 .047 27.6 58.4 88.731.1 32 .010 .020 35.6 217.6 321.7 NT 37 .038 .048 36.7 97.6 196.9 183.241 .028 .052 32.3 101.7 100.4 63.4 Mean ± SE .03 .04 30.7 116 211 187±.01 ±.01 ±2.8 ±26.8 ±57.5 ±63.4 Experimental (E. coli + LACI) 29 NT NT30.0 57.3 50.8 12.5 30 .150 .639 64.2 51.1 26.1 7.1 31 .013 .030 31.848.0 36.7 10.7 38 .034 .049 16.5 42.7 30.6 6.4 40 .059 .058 17.3 24.823.7 11.3 Mean ± SE .06 .19 32.0 44.8 33.6 9.6 ±.03 ±.129 ±8.7 ±5.5 ±4.8±1.2

[0225] TABLE 8 Weight, Sex, E. coli Dose and Survival Times of Controland LACI* Treated Baboons at +240 min** Weight Mean Dose E. coliSurvival (kg) Sex (CFU/kg × 10¹⁰) Time (hrs) Control (E. coli +excipient control) 33 6.8 M 6.22 63.5 45 7.7 F 6.29 18.0 46 9.1 M 4.9432.5 47 6.6 M 5.26 9.0 48 7.1 M 5.70 18.0 Mean 7.5 5.68 28.2 (±SE) ±0.5±.26 ±9.6 Experimental (E. coli + LACI) 34 5.2 M 5.65 58 35 7.3 M5.62 >168 36 6.8 M 4.84 >168 44 9.1 M 5.87 69 49 7.5 F 5.17 35 Mean 7.25.43 99.6 (±SE) ±0.6 ±0.19 ±28.5

[0226] The present invention has been described with reference tospecific embodiments. However, this application is intended to coverthose changes and substitutions which may be made by those skilled inthe art without departing from the spirit and the scope of the appendedclaims.

1 4 1 74 DNA Homo sapiens 1 catggctgat tctgaagaag atgaagaaca tactacgactaagacttctt ctacttcttg 60 tatgataata gtga 74 2 68 DNA Homo sapiens 2ttatcactga tactgaactg ccaccgctga aactgatgca ctatgacttg acggtggcga 60ctttgact 68 3 21 DNA Homo sapiens 3 ataacaaagc ttacatattt t 21 4 20 DNAHomo sapiens 4 atatatccat ggctgattct 20

We claim:
 1. A method of treating sepsis-associated DIC comprising:administering to a patient who has sepsis-associated DIC atherapeutically effective amount of LACI in the absence of heparin. 2.The method of claim 1 wherein said LACI comprises a first Kunitz-domainconsisting of amino acids 47-117.
 3. The method of claim 1 wherein saidLACI comprises a second Kunitz domain consisting of amino acids 118-188.4. The method of claim 1 wherein said LACI comprises a first and asecond Kunitz domain consisting of amino acids 47-188.
 5. The method ofclaim 2 wherein said LACI lacks a third domain.
 6. The method of claim 1wherein said LACI is administered in a total daily dose of 4-20 mg/kg.7. The method of claim 1 wherein said LACI is administered in a totaldaily dose of 6-10 mg/kg.
 8. The method of claim 1 wherein said LACI isadministered in a total daily-dose of 2-50 mg/kg.
 9. A method fortreating septic patients who do not have DIC, comprising: administeringto a septic patient who does not have DIC a therapeutically effectiveamount of LACI.
 10. The method of claim 9 wherein said LACI isadministered at a dose of 1 ug to 20 ug per kg.
 11. The method of claim9 wherein said LACI is administered at a dose of 20 ug to 10 mg per kg.12. The method of claim 9 wherein said LACI is administered at a dose of1 mg to 7 mg per kg.
 13. The method of claim 9 wherein said LACIcomprises a first Kunitz-domain consisting of amino acids 47-117. 14.The method of claim 9 wherein said LACI comprises a second Kunitz domainconsisting of amino acids 118-188.
 15. The method of claim 9 whereinsaid LACI comprises a first and a second Kunitz domain consisting ofamino acids 47-188.
 16. The method of claim 13 wherein said LACI lacks athird domain.
 17. A prophylactic method for decreasing the risk andseverity of sepsis comprising: administering to a patient susceptible tosepsis a prophylactically effective amount of LACI.
 18. The method ofclaim 17 wherein said LACI comprises a first Kunitz-domain consisting ofamino acids 47-117.
 19. The method of claim 17 wherein said LACIcomprises a second Kunitz domain consisting of amino acids 118-188. 20.The method of claim 17 wherein said LACI comprises a first and a secondKunitz domain consisting of amino acids 47-188.
 21. The method of claim18 wherein said LACI lacks a third domain.
 22. The method of claim 17wherein said LACI is administered at a dose of 1 ug to 20 ug per kg. 23.The method of claim 17 wherein said LACI is administered at a dose of 20ug to 10 mg per kg.
 24. The method of claim 17 wherein said LACI isadministered at a dose of 1 mg to 7 mg per kg.
 25. A method forprophylactically and therapeutically treating acute inflammation,including sepsis and septic shock, comprising administering to a patienta therapeutically effective amount of LACI.
 26. A method in accordancewith claim 25, wherein LACI is administered at a dose between 1 μg/kg to20 mg/kg.
 27. A method in accordance with claim 25, wherein LACI isadministered at a dose between 20 μg/kg to 10 mg/kg.
 28. A method inaccordance with claim 25, wherein LACI is administered at a dose between1 to 7 mg/kg.
 29. A method in accordance with claim 25 furthercomprising adding an additional agent to treat sepsis.
 30. A method inaccordance with claim 29, wherein the additional agents are selectedfrom the group consisting of antibiotics, monoclonal antibodies, andcytokine and complement inhibitors.
 31. A method in accordance withclaim 25, wherein LACI is chemically conjugated to a polymer consistingessentially of PEG or POG.
 32. A method in accordance with claim 25,wherein LACI includes a fragment or a hybrid molecule thereof.
 33. Amethod in accordance with claim 25, wherein sepsis is treated byinducing native LACI.
 34. A method for treating a disease state in whichTNF, IL-1 and other cytokines up-regulate tissue factor comprisingadministering LACI.
 35. A method in accordance with claim 34, whereinthe disease state is chronic or acute inflammation.
 36. A method inaccordance with claim 34, wherein the in vivo circulating concentrationof IL-6 is reduced.
 37. A method for treating inflammation comprisingadministering to a patient a therapeutically effective amount of LACI ora fragment thereof.
 38. A method in accordance with claim 37, whereinLACI is administered at a dose between 1 μg/kg to 20 mg/kg.
 39. A methodin accordance with claim 37, wherein LACI is administered at a dosebetween 20 μg/kg to 10 mg/kg.
 40. A method in accordance with claim 37,wherein LACI is administered at a dose between 1 to 7 mg/kg.